Polynucleotides and polypeptide sequences involved in the process of bone remodeling

ABSTRACT

This invention relates, in part, to unique and newly identified genetic polynucleotides involved in the process of bone remodeling; variants and derivatives of the polynucleotides and corresponding polypeptides; uses of the polynucleotides, polypeptides, variants and derivatives; and methods and compositions for the amelioration of symptoms caused by bone remodeling disorders. Disclosed in particular are, the isolation and identification of polynucleotides, polypeptides, variants and derivatives involved in osteoclast activity, validation of the identified polynucleotides for their potential as therapeutic targets and use of the polynucleotides, polypeptides, variants and derivatives for the amelioration of disease states and research purposes.

FIELD OF THE INVENTION

This invention relates, in part, to unique and newly identified geneticpolynucleotides involved in the process of bone remodeling; variants andderivatives of the polynucleotides and corresponding polypeptides; usesof the polynucleotides, polypeptides, variants and derivatives; methodsand compositions for the amelioration of symptoms caused by boneremodeling disorders, including but not limited to osteoporosis,osteopenia, osteomalacia, hyperparathyroidism, hypothyroidism,hyperthyroidism, hypogonadism, thyrotoxicosis, systemic mastocytosis,adult hypophosphatasia, hyperadrenocorticism, osteogenesis imperfecta,Paget's disease, Cushing's disease/syndrome, Tumer syndrome, Gaucherdisease, Ehlers-Danlos syndrome, Marfan's syndrome, Menkes' syndrome,Fanconi's syndrome, multiple myeloma, hypercalcemia, hypocalcemia,arthritides, periodontal disease, rickets (including vitamin Ddependent, type I and II, and x-linked hypophosphatemic rickets),fibrogenesis imperfecta ossium, osteosclerotic disorders such aspycnodysostosis and damage caused by macrophage-mediated inflammatoryprocesses.

In particular, this invention relates to polynucleotide expressionprofiles of active osteoclasts, the isolation and identification ofpolynucleotides, polypeptides, variants and derivatives involved inosteoclast activity, validation of the identified polynucleotides fortheir potential as therapeutic targets and use of the polynucleotides,polypeptides, variants and derivatives for the amelioration of diseasestates and research purposes, as well as in diagnosis of disease statesor in the predisposition to develop same.

BACKGROUND OF THE INVENTION

Bone is a dynamic connective tissue comprised of functionally distinctcell populations required to support the structural, mechanical andbiochemical integrity of bone and the human body's mineral homeostasis.The principal cell types involved include, osteoblasts responsible forbone formation and maintaining bone mass, and osteoclasts responsiblefor bone resorption. Osteoblasts and osteoclasts function in a dynamicprocess termed bone remodeling. The development and proliferation ofthese cells from their progenitors is governed by networks of growthfactors and cytokines produced in the bone microenvironment as well asby systemic hormones. Bone remodeling is ongoing throughout the lifetimeof the individual and is necessary for the maintenance of healthy bonetissue and mineral homeostasis. The process remains largely inequilibrium and is governed by a complex interplay of systemic hormones,peptides and downstream signalling pathway proteins, local transcriptionfactors, cytokines, growth factors and matrix remodeling genes.

Any interference or imbalance arising in the bone remodeling process canproduce skeletal disease, with the most common skeletal disorderscharacterized by a net decrease in bone mass. A primary cause of thisreduction in bone mass is an increase in osteoclast number and/oractivity. The most common such disease, and perhaps the most well known,is osteoporosis occurring particularly in women after the onset ofmenopause. In fact osteoporosis is the most significant underlying causeof skeletal fractures in late middle-aged and elderly women. Whileestrogen deficiency has been strongly implicated as a factor inpostmenopausal osteoporosis, there is longstanding evidence thatremodeling is a locally controlled process being that it takes place indiscrete packets throughout the skeleton as first described by Frostover forty years ago (Frost H. M. 1964).

Since bone remodeling takes place in discrete packets, locally producedhormones and enzymes may be more important than systemic hormones forthe initiation of bone resorption and the normal remodeling process.Such local control is mediated by osteoblasts and osteoclasts in themicroenvironment in which they operate. For example, osteoclasts attachto the bone matrix and form a separate compartment between themselvesand the bone surface delimited by a sealing zone formed by a ring ofactin surrounding the ruffled border. Multiple small vesicles transportenzymes toward the bone matrix and internalize partially digested bonematrix. The microenvironment within the sealing zone is rich with thepresence of lysosomal enzymes and is highly acidic compared to thenormal physiological pH of the body. The ruffled border membrane alsoexpresses RANK, the receptor for RANKL, and macrophage-colonystimulating factor (M-CSF) receptor, both of which are responsible forosteoclast differentiation, as well as the calcitonin receptor capableof rapidly inactivating the osteoclast (Baron, R. 2003).

In a complex pattern of inhibition and stimulation not yet fullyunderstood, growth hormone, insulin-like growth factor-1, the sexsteroids, thyroid hormone, calciotrophic hormones such as PTH andprostaglandin E2, various cytokines, such as interleukin-1 beta,interleukin-6, and tumour necrosis factor-alpha, and1,25-dihydroxyvitamin D (calcitriol) act co-ordinately in the boneremodeling process (Jilka et al. 1992; Poli et al. 1994; Srivastava etal. 1998; de Vemejoul 1996).

Thus, it stands to reason that the unique local environments created bythese specialized cells is due to the expression of either uniquegenetic sequences not expressed in other tissues and/or splice variantsof polynucleotides and polypeptides expressed in other tissues. Theisolation and identification of polynucleotides, polypeptides and theirvariants and derivatives specific to osteoclast activity will permit aclearer understanding of the remodeling process and offer tissuespecific therapeutic targets for the treatment of disease states relatedto bone remodeling.

Many diseases linked to bone remodeling are poorly understood, generallyuntreatable or treatable only to a limited extent. For example,osteoarthritis is difficult to treat as there is no cure and treatmentfocuses on relieving pain and preventing the affected joint frombecoming deformed. Non-steroidal anti-inflammatory drugs (NSAIDs) aregenerally used to relieve pain.

Another example is osteoporosis where the only current medicationsapproved by the FDA for use in the United States are the anti-resorptiveagents that prevent bone breakdown. Estrogen replacement therapy is oneexample of an anti-resorptive agent. Others include alendronate(Fosamax—a biphosphonate anti-resorptive), risedronate (Actonel—abisphosphonate anti-resorptive), raloxifene (Evista—selective estrogenreceptor modulator (SERM)), calcitonin (Calcimar—a hormone), andparathyroid hormone/teriparatide (Forteo—a synthetic version of thehuman hormone, parathyroid hormone, which helps to regulate calciummetabolism).

Bisphosphonates such as alendronate and risedronate bind permanently tothe surface of bone and interfere with osteoclast activity. This allowsthe osteoblasts to outpace the rate of resorption. The most common sideeffects are nausea, abdominal pain and loose bowel movements. However,alendronate is reported to also cause irritation and inflamation of theesophagus, and in some cases, ulcers of the esophagus. Risedronate ischemically different from alendronate and has less likelihood of causingesophagus irritation. However, certain foods, calcium, iron supplements,vitamins and minerals, or antacids containing calcium, magnesium, oraluminium can reduce the absorption of risedronate, thereby resulting inloss of effectiveness.

The most common side effect of Raloxifen and other SERMS (such asTamoxifen) are hot flashes. However, Raloxifene and other hormonereplacement therapies have been shown to increase the risk of bloodclots, including deep vein thrombosis and pulmonary embolism,cardiovascular disease and cancer.

Calcitonin is not as effective in increasing bone density andstrengthening bone as estrogen and the other anti-resorptive agents.Common side effects of either injected or nasal spray calcitonin arenausea and flushing. Patients can develop nasal irritations, a runnynose, or nosebleeds. Injectable calcitonin can cause local skin rednessat the site of injection, skin rash, and flushing.

A situation demonstrative of the link between several disorders ordisease states involving bone remodeling is that of the use ofetidronate (Didronel) first approved by the FDA to treat Paget'sdisease. Paget's disease is a bone disease characterized by a disorderlyand accelerated remodeling of the bone, leading to bone weakness andpain. Didronel has been used ‘off-label’ and in some studies shown toincrease bone density in postmenopausal women with establishedosteoporosis. It has also been found effective in preventing bone lossin patients requiring long-term steroid medications (such as Prednisoneor Cortisone). However, high dose or continuous use of Didronel cancause another bone disease called osteomalacia. Like osteoporosis,osteomalacia can lead to weak bones with increased risk of fractures.Because of osteomalacia concerns and lack of enough studies yetregarding reduction in the rate of bone fractures, the United States FDAhas not approved Didronel for the treatment of osteoporosis.

Osteoporosis therapy has been largely focused on antiresorptive drugsthat reduce the rate of bone loss but emerging therapies show promise inincreasing bone mineral density instead of merely maintaining it orslowing its deterioration. The osteoporosis early stage pipelineconsists largely of drug candidates in new therapeutic classes, inparticular cathepsin K inhibitors, osteoprotegerin and calcilytics aswell as novel bisphosphonates. Some of these are examples where noveldrugs exploiting genomics programs are being developed based on a deeperunderstanding of bone biology and have the potential to change the faceof treatment of bone disorders in the long term.

The present invention satisfies a need in the art. There thus remains aneed to better understand the bone remodeling process and to provide newcompositions that are useful for the diagnosis, prognosis, treatment,prevention and evaluation of therapies for bone remodeling andassociated disorders. A method for analysing polynucleotide expressionpatterns has been developed and applied to identify polynucleotides,polypeptides, variants and derivatives specifically involved in boneremodeling.

The present invention seeks to meet these and other needs.

The present description refers to a number of documents, the content ofwhich is herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The present invention relates to polynucleotides comprising sequencesinvolved in the process of bone remodeling including their open readingframe, substantially identical sequences, substantially complementarysequences and fragments thereof.

The present invention relates to polypeptide comprising sequencesinvolved in the process of bone remodeling including biologically activeanalogs and biologically active fragments thereof.

The present invention also relates to compositions that are useful forthe diagnosis, prognosis, treatment, prevention and/or evaluation oftherapies for bone remodeling and associated disorders.

In addition, the present invention relates to a method for analyzingpolynucleotide expression patterns, and applied to identifypolynucleotides, polypeptides, variants and derivatives specificallyinvolved in bone remodeling.

Furthermore, the present invention relates to polynucleotide andpolypeptide sequences, variants and derivatives thereof which have beenvalidated as potential therapeutic targets.

The identification of gene products involved in regulating osteoclastdifferentiation and function has led to the discovery of novel targetsfor the development of new and specific therapies of disease statescharacterized by abnormal bone remodeling.

The present invention relates to polynucleotide expression profiles ofosteoclasts, the isolation and identification of polynucleotides, theircorresponding polypeptides, variants and derivatives involved inosteoclast activity, validation of these identified elements for theirpotential as therapeutic targets and use of said polynucleotides,polypeptides, variants and derivatives for the amelioration of diseasestates.

It is an object of the present invention to provide polynucleotides andrelated polypeptides that have been isolated and identified. Morespecifically, the invention provides polynucleotides comprising any oneof SEQ. ID. NOs:1 to 57 or 83 to 89, their coding sequence (open readingframe) and related polypeptides comprising any one of SEQ ID NO.: 93 to155 which have been shown to be upregulated in a highly specific fashionin osteoclasts.

The present invention more particularly relates to polynucleotides,their coding sequence (open reading frame), and related polypeptides,which have been demonstrably shown to be necessary or crucial forosteoclast differentiation (e.g. SEQ. ID. NOs:1 to 7, 88 and 89).

Of the polynucleotides (e.g. SEQ. ID. NOs:8 to 56) whose gene expressionis upregulated, 37 were tested in the model using siRNA for biologicalvalidation leaving 12 still to be tested, 28 does not appear tophenotypically perturb osteoclast differentiation in the model usedwhereas 9 did (SEQ ID NO.:16, SEQ ID NO.:19, SEQ ID NO.:21, SEQ IDNO.:24, SEQ ID NO.:29, SEQ ID NO.:31, SEQ ID NO.:37 and SEQ ID NO.:42).However, a more discrete effect not phenotypically measurable cannot beruled out for those 28. Without being limited to a particular model,this may be due in part to non-functional siRNA and/or to their roles inthe downstream bone remodeling activities of osteoclasts. For example,polynucleotides for cathepsin K (CTSK) and matrix metalloproteinase 9(MMP-9) are well known markers which are essential for osteoclastactivities in bone remodelling but are not required for osteoclastdifferentiation. NSEQ refers generally to polynucleotide sequences ofthe present invention and includes for example, SEQ. ID. NOs:1 to 56 and83 to 89, whereas PSEQ refers generally to polypeptide sequences of thepresent invention and includes, for example, SEQ ID NO.:93 to 99 and 101to 155. Of course it will be understood that NSEQ also encompassespolynucleotide sequences which are designed or derived from SEQ. ID.NOs:1 to 57 and 83 to 89 and more particularly from their codingsequence. Non-limiting examples of such sequences are disclosed herein(e.g. SEQ ID Nos 64-82 and 90).

The present invention also provides a method of using a polynucleotideselected from SEQ ID NO's 1 to 57 and 83 to 89 and more particularlytheir coding sequence and encoded polypeptides thereof to screen alibrary of molecules or compounds (e.g. DNA molecules, RNA molecules,PNAs, mimetics and proteins) to identify or purify a ligand whichspecifically binds the polynucleotide by combining a polynucleotide witha library of molecules or compounds under conditions to allow specificbinding, and detecting specific binding, thereby identifying orpurifying a ligand which specifically binds the polynucleotide.

The present invention relates in one aspect thereof to an isolatedpolynucleotide sequence having at least from about 80% to about 100%(e.g., 80%, 90%, 95%, etc.) nucleic acid sequence identity to apolynucleotide sequence selected from the group consisting ofpolynucleotides comprising (a) any one of a SEQ. ID. NOs:1 to 57 and 83to 89; (b) an open reading frame of (a); (c) a full complement of (a) or(b), and; (d) a fragment of any one of (a) to (c).

Complements of the isolated polynucleotide sequence encompassed by thepresent invention may be those, for example, which hybridize under highstringency conditions to any of the nucleotide sequences in (a), or (b).The high stringency conditions may comprise, for example, ahybridization reaction at 65° C. in 5×SSC, 5× Denhardt's solution, 1%SDS, and 100 μg/ml denatured salmon sperm DNA.

In accordance with the present invention, the polynucleotide sequencemay be used, for example, in the treatment of diseases or disordersinvolving bone remodelling.

Fragments of polynucleotides may be used, for example, as probes fordetermining the presence of the isolated polynucleotide (or itscomplement or fragments thereof) in a sample, cell, tissue, etc. forexperimental purposes or for the purpose of diagnostic of a diseases ordisorders involving bone remodelling.

The present invention also relates to a combination comprising aplurality of polynucleotides (substantially purified and/or isolated)that may be co-expressed with one or more genes known to be involved inbone remodelling, the plurality of polynucleotides may be selected, forexample, from the group consisting of a polynucleotide comprising (a)any one of SEQ. ID. NOs:1 to 57, 83 to 89; (b) an open reading frame(a); (c) a full complement of (a) or (b); (d) a sequence that hybridizesunder high stringency conditions to any one of the nucleotide sequencesin (a), or (b) and; (e) fragments of (a), (b), (c) or (d).

The present invention further relates to a polynucleotide encoding anyone of the polypeptides described herein. In accordance with the presentinvention, the polynucleotide (RNA, DNA, etc.) may encode a polypeptidewhich may be selected from the group consisting of any one of SEQ IDNO.:93 to 155, analogs or fragments thereof (e.g., biologically activefragments, immunologically active fragments, etc.).

The present invention also relates to an isolated nucleic acid moleculecomprising the polynucleotides of the present invention, operativelylinked to a nucleotide sequence encoding a heterologous polypeptidethereby encoding a fusion polypeptide.

The invention further relates to a polypeptide encoded by apolynucleotide of SEQ. ID. NOs:1 to 56 or 83 to 89 and more particularlyfrom the open reading frame of any one of SEQ. ID. NOs:1 to 56 or 83 to89, or a portion thereof, comprising the product of a gene that isco-expressed with one or more genes known to be involved in boneremodeling.

The invention additionally relates to the use of the polypeptide or aportion thereof to screen a library of molecules or compounds (DNAmolecules, RNA molecules, PNAs, mimetics, proteins, agonists,antagonists, and antibodies) to identify or purify at least one ligandwhich specifically binds the polypeptide by combining the polypeptide ora portion thereof with the library of molecules or compounds underconditions to allow specific binding, and detecting specific bindingbetween the polypeptide and ligand, thereby identifying or purifying aligand which specifically binds the polypeptide.

Isolated naturally occurring allelic variant are also encompassed by thepresent invention as well as synthetic variants (e.g., made byrecombinant DNA technology or by chemical synthesis, etc.) such asbiologically active variant which may comprise one or more conservativeamino acid substitutions (compared to a naturally occurringpolypeptide).

The present invention, further provides a vector (mammalian, bacterial,viral, etc.) comprising the polynucleotides described herein orfragments thereof, such as an expression vector. The vector may furthercomprise a nucleic acid sequence which may help in the regulation ofexpression of the polynucleotide and/or a nucleotide sequence encoding atag (e.g., affinity tag; HA, GST, His etc.).

In accordance with the present invention, an expression vector maycomprise, for example, the following operatively linked elements:

-   -   a) a transcription promoter;    -   b) a polynucleotide segment (which may comprise an open reading        frame); and    -   c) a transcription terminator.

The invention also relates to an expression vector comprising apolynucleotide described herein, a host cell transformed with theexpression vector and a method for producing a polypeptide of thepresent invention.

More particularly, the present invention therefore provides a cell whichmay be genetically engineered to contain and/or to express thepolynucleotide (including complements and fragments) and/or polypeptidesof the present invention. The cell may be, for example, a mammaliancell, an insect cell, a bacteria cell, etc.

The present invention, therefore provides a host cell which may comprisea vector as described herein. The cell may be, for example, mammaliancell, an insect cell, a bacteria, etc. The cell may be able to expressor expresses a polypeptide encoded by the polynucleotide describedherein.

Methods of producing the polypeptides of the present inventionencompassed herewith includes for example, culturing the cell inconditions allowing the expression of the polypeptide. The polypeptidemay be recovered, for example, from cell lysate or from the cellsupernatant.

The present invention also relates to a method of using a polynucleotidesequence described herein to screen a library of molecules or compoundsincluding but not limited to, DNA molecules, RNA molecules, PNAs(peptide nucleic acids), peptides, ribozymes, antibodies, agonists,antagonists, immunoglobulins, inhibitors, proteins includingtranscription factors, enhancers, repressors, and drugs and the likewhich regulate the activity of the selected polynucleotide sequence in abiological system, to identify or purify a ligand which may specificallybind the polynucleotide by combining a polynucleotide with a library ofmolecules or compounds under conditions which may allow specificbinding, and detecting specific binding, thereby identifying orpurifying a ligand which may specifically bind the polynucleotide.

The antagonist, agonist, ligand thus identified may be used in thetreatment of bone remodelling diseases or disorders.

The invention relates to the use of at least one polynucleotidecomprising any one of SEQ. ID. NOs:1 to 57 and/or 83 to 89, their codingsequence, substantially identical sequences, substantially complementarysequences and fragments thereof on an array and for the use of thatarray in a method for diagnosing a bone remodeling disease or disorderby hybridizing the array with a patient sample under conditions to allowcomplex formation, detecting complex formation, and comparing the amountof complex formation in the patient sample to that of standards fornormal and diseased tissues wherein the complex formation in the patientsample indicates the presence of a bone remodeling disease or disorder.Of course, the use of a polynucleotide of the present invention in adiagnosis method is not dependent exclusively by way of an assay. Thesequence or sequences may be used in conventionally used diagnosismethods known in the art.

The present invention also relates to a method of ameliorating boneremodelling disease or disorder symptoms, or for inhibiting or delayingbone disease or disorder, the method may comprise: contacting a compoundcapable of specifically inhibiting activity or expression of apolynucleotide sequence described herein or a polypeptide describedherein, in osteoclasts so that symptoms of the bone remodelling diseaseor disorder may be ameliorated, or the disease or disorder may beprevented, delayed or lowered.

The present invention further relates to a method for ameliorating boneremodelling disease or disorder symptoms, or for inhibiting or delayingbone disease or disorder, the method may comprise: contacting a compoundcapable of specifically promoting activity or expression of apolynucleotide sequence described herein or a polypeptide describedherein, in osteoclasts so that symptoms of the bone remodelling diseaseor disorder may be ameliorated, or the disease or disorder may beprevented, delayed or lowered.

The present invention also relates to a method of treating a conditionin a mammal characterized by a deficiency in, or need for, bone growthor replacement and/or an undesirable level of bone resorption, whichmethod may comprise administering to a mammalian' subject in need ofsuch treatment an effective amount of a suitable compound describedherein.

The present invention further relates to a method of using apolynucleotide sequence described herein, a polypeptide described hereinon an array and for the use of the array in a method for diagnosing abone remodelling disease or disorder by hybridizing the array with apatient sample under conditions to allow complex formation, detectingcomplex formation, and comparing the amount of complex formation in thepatient sample to that of standards for normal and diseased tissueswherein the complex formation in the patient sample may indicate thepresence of a bone remodelling disease or disorder.

In accordance with the present invention the isolated polynucleotidesequence described herein, the antagonist described herein, the liganddescribed herein, or the method described herein, may be used fordiseases or disorders which may be selected from the group consistingof, but not limited to, osteoporosis, osteopenia, osteomalacia,hyperparathyroidism, hyperthyroidism, hypogonadism, thyrotoxicosis,systemic mastocytosis, adult hypophosphatasia, hyperadrenocorticism,osteogenesis imperfecta, Paget's disease, Cushing's disease/syndrome,Turner syndrome, Gaucher disease, Ehlers-Danlos syndrome, Marfan'ssyndrome, Menkes' syndrome, Fanconi's syndrome, multiple myeloma,hypercalcemia, hypocalcemia, arthritides, periodontal disease, rickets(including vitamin D dependent, type I and II, and x-linkedhypophosphatemic rickets), fibrogenesis imperfecta ossium,osteosclerotic disorders such as pycnodysostosis and damage caused bymacrophage-mediated inflammatory processes.

In accordance with the present invention, the method of administrationmay be selected from, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, transdermal, subcutaneous, intraperitoneal,intranasal, enteral, topical, sublingual, or rectal means.

In accordance with the present invention, the polynucleotide sequencedescribed herein may be used for somatic cell gene therapy or for stemcell gene therapy.

The invention also relates to a pharmaceutical composition comprising apolynucleotide described herein, a polypeptide encoded by the selectedpolynucleotide, a portion thereof, a ligand (agonist or antagonist)identified or purified using a selected polynucleotide or a polypeptideencoded by the selected polynucleotide, or a portion thereof, whichmodulates the activity (activation, enhancement or inhibition) of theselected polynucleotide or a polypeptide encoded thereby, a portionthereof, and a suitable pharmaceutical carrier.

Additionally, the invention relates to products, compositions, processesand methods that comprises a polynucleotide described herein, apolypeptide encoded by the polynucleotides, a portion thereof, theirvariants or derivatives, for research, biological, clinical andtherapeutic purposes.

The NSEQs and PSEQs may be used in diagnosis, prognosis, treatment,prevention, and selection and evaluation of therapies for diseases anddisorders involving bone remodeling including, but not limited to,osteoporosis, osteopenia, osteomalacia, hyperparathyroidism,hyperthyroidism, hyperthyroidism, hypogonadism, thyrotoxicosis, systemicmastocytosis, adult hypophosphatasia, hyperadrenocorticism, osteogenesisimperfecta, Paget's disease, Cushing's disease/syndrome, Turnersyndrome, Gaucher disease, Ehlers-Danlos syndrome, Marfan's syndrome,Menkes' syndrome, Fanconi's syndrome, multiple myeloma, hypercalcemia,hypocalcemia, arthritides, periodontal disease, rickets (includingvitamin D dependent, type I and II, and x-linked hypophosphatemicrickets), fibrogenesis imperfecta ossium, osteosclerotic disorders suchas pycnodysostosis and damage caused by macrophage-mediated inflammatoryprocesses.

Use of NSEQ as a Screening Tool

The polynucleotides obtained by the present invention may be used todetect and isolate expression products, for example, mRNA, complementaryDNAs (cDNAs) and proteins derived from or homologous to the NSEQs. Inone embodiment, the expression of mRNAs homologous to the NSEQs of thepresent invention may be detected, for example, by hybridizationanalysis, reverse transcription and in vitro nucleic acid amplificationmethods. Such procedures permit detection of mRNAs in a variety oftissue types or at different stages of development. The subject nucleicacids which are expressed in a tissue-specific or adevelopmental-stage-specific manner are useful as tissue-specificmarkers or for defining the developmental stage of a sample of cells ortissues that may define a particular disease state. One of skill in theart may readily adapt the NSEQs for these purposes.

Those skilled in the art will also recognize that the NSEQs, and itsexpression products such as cDNA nucleic acids and genomic DNA may beused to prepare short oligonucleotides sequences. For example,oligonucleotides having ten to twelve nucleotides or more may beprepared which hybridize specifically to the present NSEQs and cDNAs andallow detection, identification and isolation of unique nucleicsequences by hybridization. Sequences of for example, at least 15-20nucleotides may be used and selected from regions that lack homology toother known sequences. Sequences of 20 or more nucleotides that lacksuch homology show an increased specificity toward the target sequence.Useful hybridization conditions for probes and primers are readilydeterminable by those of skill in the art. Stringent hybridizationconditions encompassed herewith are those that may allow hybridizationof nucleic acids that are greater than 90% homologous but which mayprevent hybridization of nucleic acids that are less than 70%homologous. The specificity of a probe may be determined by whether itis made from a unique region, a regulatory region, or from a conservedmotif. Both probe specificity and the stringency of diagnostichybridization or amplification (maximal, high, intermediate, or low)reactions may be determined whether the probe identifies exactlycomplementary sequences, allelic variants, or related sequences. Probesdesigned to detect related sequences may have at least 50% sequenceidentity to any of the selected polynucleotides.

It is to be understood herein that the NSEQs (substantially identicalsequences and fragments thereof) may hybridize to a substantiallycomplementary sequence found in a test sample. Additionally, a sequencesubstantially complementary to NSEQ may bind a NSEQ found in a testsample.

Skilled practitioners will also recognize that the NSEQs and PSEQs maybe used to screen a library of molecules for specific binding affinity.Typical assays may be used to screen a library of DNA molecules, RNAmolecules, PNAs (peptide nucleic acids), peptides, ribozymes,antibodies, agonists, antagonists, immunoglobulins, inhibitors, proteinsincluding transcription factors, enhancers, repressors, and drugs andthe like which regulate the activity of the selected polynucleotidesequence in a biological system. Typical assays may involve providing alibrary of molecules, combining the polynucleotide sequence or afragment thereof with the library of molecules under conditions suitableto allow specific binding, and detecting specific binding to identify orpurify, at least one molecule (ligand) which may specifically bind thepolynucleotide sequence. One of skill in the art may readily adapt theNSEQs for these purposes.

Those of skill in the art may readily label the NSEQs and PSEQs bystandard methods to add them to a sample from a subject under conditionsfor the formation and detection of hybridization complexes. Afterincubation the sample may be washed, and the signal associated withhybrid complex formation may be quantified and compared with a standardor normal value. Standard or normal values may be derived from anycontrol sample, typically one that may be free of a suspect disease. Ifthe amount of signal in the subject sample is altered in comparison tothe standard value, then the presence of altered levels of expression inthe sample may indicate the presence of the disease. Qualitative andquantitative methods for comparing the hybridization complexes formed insubject samples with previously established standards are well known inthe art.

Furthermore, a probe may be labelled by any procedure known in the art,for example by incorporation of nucleotides linked to a “reportermolecule”. A “reporter molecule”, as used herein, may be a molecule thatprovides an analytically identifiable signal allowing detection of ahybridized probe. Detection may be either qualitative or quantitative.Commonly used reporter molecules include fluorophores, enzymes, biotin,chemiluminescent molecules, bioluminescent molecules, digoxigenin,avidin, streptavidin or radioisotopes. Commonly used enzymes includehorseradish peroxidase, alkaline phosphatase, glucose oxidase andβ-galactosidase, among others. Enzymes may be conjugated to avidin orstreptavidin for use with a biotinylated probe. Similarly, probes may beconjugated to avidin or streptavidin for use with a biotinylated enzyme.Incorporation of a reporter molecule into a DNA probe may be by anymethod known to the skilled artisan, for example by nick translation,primer extension, random oligo priming, by 3′ or 5′ end labeling or byother means. In addition, hybridization probes include the cloning ofnucleic acid sequences into vectors for the production of mRNA probes.Such vectors are known in the art, are commercially available, and maybe used to synthesize RNA probes in vitro. The labelled polynucleotidesequences may be used in Southern or northern analysis, dot blot, orother membrane-based technologies; in PCR technologies; and in microarrays utilizing samples from subjects to detect altered expression.Oligonucleotides useful as probes for screening of samples byhybridization assays or as primers for amplification may be packagedinto kits. Such kits may contain the probes or primers in a pre-measuredor predetermined amount, as well as other suitably packaged reagents andmaterials needed for the particular hybridization or amplificationprotocol.

In another embodiment, the invention entails a substantially purifiedpolypeptide encoded by the polynucleotides of NSEQs, polypeptide analogsor polypeptide fragments thereof. The polypeptides whether in apremature, mature or fused form, may be isolated from lysed cells, orfrom the culture medium, and purified to the extent needed for theintended use. One of skill in the art may readily purify these proteins,polypeptides and peptides by any available procedure. For example,purification may be accomplished by salt fractionation, size exclusionchromatography, ion exchange chromatography, reverse phasechromatography, affinity chromatography and the like.

The invention further provides for a polypeptide encoded by thepolynucleotides of NSEQs, or a portion thereof, comprising the productof a gene that is co-expressed with one or more genes known to beinvolved in bone remodeling. The invention additionally provides for theuse of the polypeptide or a portion thereof to screen a library ofmolecules or compounds (DNA molecules, RNA molecules, PNAs, mimetics,proteins, agonists, antagonists, and antibodies) to identify or purifyat least one ligand which specifically binds the polypeptide bycombining the polypeptide or a portion thereof with the library ofmolecules or compounds under conditions to allow specific binding, anddetecting specific binding between the polypeptide and ligand, therebyidentifying or purifying a ligand which specifically binds thepolypeptide. One of skill in the art may readily adapt the NSEQs forthese purposes.

The portion of a polypeptide employed in such screening may be free insolution, affixed to an abiotic or biotic substrate or locatedintra-cellularly. Specific binding between the polypeptide and themolecule may be measured. The assay may be used to screen a library ofDNA molecules, RNA molecules, PNAs, peptides, mimetics, ribozymes,antibodies, agonists, antagonists, immunoglobulins, inhibitors,peptides, polypeptides, drugs and the like, which may specifically bindthe polypeptide. Many such assay methodologies are well known in the artand may be readily adapted by a skilled practitioner.

Use of NSEQ for Development of an Expression System

In order to express a biologically active polypeptide, NSEQ, orderivatives thereof, may be inserted into an expression vector, i.e., avector that contains the elements for transcriptional and translationalcontrol of the inserted coding sequence in a particular host. Theseelements include regulatory sequences, such as enhancers, constitutiveand inducible promoters, and 5′ and 3′ un-translated regions. Methodsthat are well known to those skilled in the art may be used to constructsuch expression vectors. These methods include in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination.

A variety of expression vector/host cell systems known to those of skillin the art may be utilized to express NSEQ. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith baculovirus vectors; plant cell systems transformed with viral orbacterial expression vectors; or animal cell systems. For long-termproduction of recombinant proteins in mammalian systems, stableexpression in cell lines may be effected. For example, NSEQ may betransformed into cell lines using expression vectors that may containviral origins of replication and/or endogenous expression elements and aselectable or visible marker gene on the same or on a separate vector.The invention is not to be limited by the vector or host cell employed.

In general, host cells that contain NSEQ and that express a polypeptideencoded by the NSEQ, or a portion thereof, may be identified by avariety of procedures known to those of skill in the art. Theseprocedures include, but are not limited to, DNA-DNA or DNA-RNAhybridizations, PCR amplification, and protein bioassay or immunoassaytechniques that include membrane, solution, or chip based technologiesfor the detection and/or quantification of nucleic acid or amino acidsequences. Immunological methods for detecting and measuring theexpression of polypeptides using either specific polyclonal ormonoclonal antibodies are known in the art. Examples of such techniquesinclude enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays(RIAs), and fluorescence activated cell sorting (FACS). Those of skillin the art may readily adapt these methodologies to the presentinvention.

The present invention additionally relates to a bioassay for evaluatingcompounds as potential antagonists of the polypeptide described herein,the bioassay may comprise:

-   -   a) culturing test cells in culture medium containing increasing        concentrations of at least one compound whose ability to inhibit        the action of a polypeptide described herein is sought to be        determined, wherein the test cells may contain a polynucleotide        sequence described herein in a form having improved        trans-activation transcription activity, relative to wild-type        polynucleotide, and comprising a response element operatively        linked to a reporter gene; and thereafter    -   b) monitoring in the cells the level of expression of the        product of the reporter gene as a function of the concentration        of the potential antagonist compound in the culture medium,        thereby indicating the ability of the potential antagonist        compound to inhibit activation of the polypeptide encoded by,        the polynucleotide sequence described herein.

The present invention further relates to a bioassay for evaluatingcompounds as potential agonists for a polypeptide encoded by thepolynucleotide sequence described herein, the bioassay may comprise:

-   -   a) culturing test cells in culture medium containing increasing        concentrations of at least one compound whose ability to promote        the action of the polypeptide encoded by the polynucleotide        sequence described herein is sought to be determined, wherein        the test cells may contain a polynucleotide sequence described        herein in a form having improved trans-activation transcription        activity, relative to wild-type polynucleotide, and comprising a        response element operatively linked to a reporter gene; and        thereafter    -   b) monitoring in the cells the level of expression of the        product of the reporter gene as a function of the concentration        of the potential agonist compound in the culture medium, thereby        indicating the ability of the potential agonist compound to        promote activation of a polypeptide encoded by the        polynucleotide sequence described herein.

Host cells transformed with NSEQ may be cultured under conditions forthe expression and recovery of the polypeptide from cell culture. Thepolypeptide produced by a transgenic cell may be secreted or retainedintracellularly depending on the sequence and/or the vector used. Aswill be understood by those of skill in the art, expression vectorscontaining NSEQ may be designed to contain signal sequences that directsecretion of the polypeptide through a prokaryotic or eukaryotic cellmembrane. Due to the inherent degeneracy of the genetic code, other DNAsequences that encode substantially the same or a functionallyequivalent amino acid sequence may be produced and used to express thepolypeptide encoded by NSEQ. The nucleotide sequences of the presentinvention may be engineered using methods generally known in the art inorder to alter the nucleotide sequences for a variety of purposesincluding, but not limited to, modification of the cloning, processing,and/or expression of the gene product. DNA shuffling by randomfragmentation and PCR reassembly of gene fragments and syntheticoligonucleotides may be used to engineer the nucleotide sequences. Forexample, oligonucleotide-mediated site-directed mutagenesis may be usedto introduce mutations that create new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, and so forth.

In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed polypeptide in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing, which cleaves a “prepro” form of thepolypeptide, may also be used to specify protein targeting, folding,and/or activity. Different host cells that have specific cellularmachinery and characteristic mechanisms for post-translationalactivities (e.g., CHO, HeLa, MDCK, HEK293, and W138) are availablecommercially and from the American Type Culture Collection (ATCC) andmay be chosen to ensure the correct modification and processing of theexpressed polypeptide.

Those of skill in the art will readily appreciate that natural,modified, or recombinant nucleic acid sequences may be ligated to aheterologous sequence resulting in translation of a fusion polypeptidecontaining heterologous polypeptide moieties in any of theaforementioned host systems. Such heterologous polypeptide moieties mayfacilitate purification of fusion polypeptides using commerciallyavailable affinity matrices. Such moieties include, but are not limitedto, glutathione S-transferase (GST), maltose binding protein,thioredoxin, calmodulin binding peptide, 6-His (His), FLAG, c-myc,hemaglutinin (HA), and monoclonal antibody epitopes.

In yet a further aspect, the present invention relates to an isolatedpolynucleotide which may comprise a nucleotide sequence encoding afusion protein, the fusion protein may comprise a fusion partner fusedto a peptide fragment of a protein encoded by, or a naturally occurringallelic variant polypeptide encoded by, the polynucleotide sequencedescribed herein, which peptide fragment, when administered to a memberof a mammalian species, may be capable of inducing the production ofantibodies that bind specifically to the protein encoded by, or anaturally occurring allelic variant polypeptide encoded by, thepolynucleotide sequence described herein.

Those of skill in the art will also readily recognize that the nucleicacid and polypeptide sequences may be synthesized, in whole or in part,using chemical or enzymatic methods well known in the art. For example,peptide synthesis may be performed using various solid-phase techniquesand machines such as the ABI 431A Peptide synthesizer (PE Biosystems)may be used to automate synthesis. If desired, the amino acid sequencemay be altered during synthesis and/or combined with sequences fromother proteins to produce a variant protein.

Use of NSEQ as a Diagnostic Screening Tool

The skilled artisan will readily recognize that NSEQ may be used fordiagnostic purposes to determine the absence, presence, or alteredexpression (i.e. increased or decreased compared to normal) of the gene.The polynucleotides may be at least 10 nucleotides long or at least 12nucleotides long, or at least 15 nucleotides long up to any desiredlength and may comprise complementary RNA and DNA molecules, branchednucleic acids, and/or peptide nucleic acids (PNAs). In one alternative,the polynucleotides may be used to detect and quantify gene expressionin samples in which expression of NSEQ is correlated with disease. Inanother alternative, NSEQ may be used to detect genetic polymorphismsassociated with a disease. These polymorphisms may be detected in thetranscript cDNA.

The invention provides for the use of at least one polynucleotidecomprising NSEQ (e.g., an open reading frame of NSEQ, a substantiallycomplementary sequence, a substantially identical sequence, andfragments thereof) on an array and for the use of that array in a methodfor diagnosing a bone remodeling disease or disorder by hybridizing thearray with a patient sample under conditions to allow complex formation,detecting complex formation, and comparing the amount of complexformation in the patient sample to that of standards for normal anddiseased tissues wherein the complex formation in the patient sampleindicates the presence of a bone remodeling disease or disorder.

In another embodiment, the present invention provides one or morecompartmentalized kits for detection of bone resorption disease states.A first kit has a receptacle containing at least one isolated probe.Such a probe may be a nucleic acid fragment which is present/absent inthe genomic DNA of normal cells but which is absent/present in thegenomic DNA of affected cells. Such a probe may be specific for a DNAsite that is normally active/inactive but which may be inactive/activein certain cell types. Similarly, such a probe may be specific for a DNAsite that may be abnormally expressed in certain cell types. Finally,such a probe may identify a specific DNA mutation. By specific for a DNAsite is meant that the probe may be capable of hybridizing to the DNAsequence which is mutated, or may be capable of hybridizing to DNAsequences adjacent to the mutated DNA sequences. The probes provided inthe present kits may have a covalently attached reporter molecule.Probes and reporter molecules may be readily prepared as described aboveby those of skill in the art.

Use of NSEQ as a Therapeutic

One of skill in the art will readily appreciate that the expressionsystems and assays discussed above may also be used to evaluate theefficacy of a particular therapeutic treatment regimen, in animalstudies, in clinical trials, or to monitor the treatment of anindividual subject. Once the presence of disease is established and atreatment protocol is initiated, hybridization or amplification assaysmay be repeated on a regular basis to determine if the level ofexpression in the patient begins to approximate the level observed in ahealthy subject. The results obtained from successive assays may be usedto show the efficacy of treatment over a period ranging from severaldays to many years.

Therefore, in a further aspect, the present invention relates to anantibody (e.g., isolated antibody), or antigen-binding fragment thereof,that may specifically bind to a protein or polypeptide described herein.The antibody may be, for example, a monoclonal antibody or a polyclonalantibody. The antibody may originate for example, from a mouse, rat orany other mammal.

The antibody may also be a human antibody which may be obtained, forexample, from a transgenic non-human mammal capable of expressing humanIg genes. The antibody may also be a humanised antibody which maycomprise, for example, one or more complementarity determining regionsof non-human origin. It may also comprise a surface residue of a humanantibody and/or framework regions of a human antibody. The antibody mayalso be a chimeric antibody which may comprise, for example, variabledomains of a non-human antibody and constant domains of a humanantibody.

Suitable antibodies may also include, for example, an antigen-bindingfragment, an Fab fragment; an F(ab′)₂ fragment, and Fv fragment; or asingle-chain antibody comprising an antigen-binding fragment (e.g., asingle chain Fv).

The antibody of the present invention may be mutated and selected basedon an increased affinity and/or specificity for one of a polypeptidedescribed herein and/or based on a reduced immunogenicity in a desiredhost.

The antibody may further comprise a detectable label attached thereto.

The present invention further relates to a method of producingantibodies able to bind to one of a polypeptide, polypeptide fragments,or polypeptide analogs described herein, the method may comprise:

-   -   a) immunizing a mammal (e.g., mouse, a transgenic mammal capable        of producing human Ig, etc.) with a suitable amount of a desired        polypeptide or a polypeptide fragment thereof;    -   b) collecting the serum from the mammal; and    -   c) isolating the polypeptide-specific antibodies from the serum        of the mammal.

The present invention also relates to a method of producing a hybridomawhich secretes an antibody that binds to a polypeptide described herein,the method may comprise:

-   -   a) immunizing a mammal (e.g., mouse, a transgenic mammal capable        of producing human Ig, etc.) with a suitable amount of a desired        polypeptide, a polypeptide fragment or analog thereof;    -   b) obtaining lymphoid cells from the immunized animal obtained        from (a);    -   c) fusing the lymphoid cells with an immortalizing cell to        produce hybrid cells; and    -   d) selecting hybrid cells which produce antibody that        specifically binds to the polypeptide, a polypeptide fragment or        analog thereof.

The present invention further relates to a method of producing anantibody that binds to one of the polypeptide described herein, themethod may comprise:

-   -   a) synthesizing a library of antibodies on phage or ribosomes;    -   b) panning the library against a sample by bringing the phage or        ribosomes into contact with a composition comprising a        polypeptide or polypeptide fragment described herein;    -   c) isolating phage which binds to the polypeptide or polypeptide        fragment, and;    -   d) obtaining an antibody from the phage or ribosomes.

The antibody of the present invention may thus be obtained, for example,from a library (e.g., bacteriophage library) which may be prepared, forexample, by

-   -   a) extracting cells which are responsible for production of        antibodies from a host mammal;    -   b) isolating RNA from the cells of (a);    -   c) reverse transcribing mRNA to produce cDNA;    -   d) amplifying the cDNA using a (antibody-specific) primer; and    -   e) inserting the cDNA of (d) into a phage display vector or        ribosome display cassette such that antibodies are expressed on        the phage or ribosomes.

The host animal may be immunized with polypeptide and/or a polypeptidefragment and/or analog described herein to induce an immune responseprior to extracting the cells which are responsible for production ofantibodies.

The present invention also relates to a kit for specifically assaying apolypeptide described herein, the kit may comprise, for example, anantibody or antibody fragment capable of binding specifically to thepolypeptide described herein.

Further, an antagonist, agonist, or an antibody that may bindspecifically to a polypeptide encoded by the polynucleotides of NSEQ, ora portion thereof, may be administered to a subject to treat or preventdiseases or disorders associated with bone remodeling. The antagonist,antibody, or fragment may be used directly to inhibit the activity ofthe polypeptide or indirectly to deliver a therapeutic agent to cells ortissues that express the NSEQ. An immunoconjugate comprising apolypeptide-binding site of the antibody or the antagonist and atherapeutic agent may be administered to a subject in need to treat orprevent disease. The therapeutic agent may be a cytotoxic agent selectedfrom a group including, but not limited to, abrin, ricin, doxorubicin,daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicine, dihydroxy anthracin dione,actinomycin D, diphteria toxin, Pseudomonas exotoxin A and 40,radioisotopes, and glucocorticoid. Yet further, an agonist of thepolypeptide may be administered to a subject to treat or prevent adisease associated with decreased expression, longevity or activity ofNSEQ.

The present invention further contemplates antibodies that may bind tothe polypeptide encoded by the polynucleotides of NSEQ, polypeptideanalogs or portions thereof. Suitable antibodies may bind to uniqueantigenic regions or epitopes in the polypeptides, or a portion thereof.Epitopes and antigenic regions useful for generating antibodies may befound within the proteins, polypeptides or peptides by proceduresavailable to one of skill in the art. For example, short, unique peptidesequences may be identified in the proteins and polypeptides that havelittle or no homology to known amino acid sequences. Preferably theregion of a protein selected to act as a peptide epitope or antigen isnot entirely hydrophobic; hydrophilic regions are preferred becausethose regions likely constitute surface epitopes rather than internalregions of the proteins and polypeptides. These surface epitopes aremore readily detected in samples tested for the presence of the proteinsand polypeptides. Such antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric, and single chain antibodies, Fabfragments, and fragments produced by a Fab expression library. Theproduction of antibodies is well known to one of skill in the art.

Peptides may be made by any procedure known to one of skill in the art,for example, by using in vitro translation or chemical synthesisprocedures. Short peptides which provide an antigenic epitope but whichby themselves are too small to induce an immune response may beconjugated to a suitable carrier. Suitable carriers and methods oflinkage are well known in the art. Suitable carriers are typically largemacromolecules such as proteins, polysaccharides and polymeric aminoacids. Examples include serum albumins, keyhole limpet hemocyanin,ovalbumin, polylysine and the like. One of skill in the art may useavailable procedures and coupling reagents to link the desired peptideepitope to such a carrier. For example, coupling reagents may be used toform disulfide linkages or thioether linkages from the carrier to thepeptide of interest. If the peptide lacks a disulfide group, one may beprovided by the addition of a cysteine residue. Alternatively, couplingmay be accomplished by activation of carboxyl groups.

The minimum size of peptides useful for obtaining antigen specificantibodies may vary widely. The minimum size must be sufficient toprovide an antigenic epitope that is specific to the protein orpolypeptide. The maximum size is not critical unless it is desired toobtain antibodies to one particular epitope. For example, a largepolypeptide may comprise multiple epitopes, one epitope beingparticularly useful and a second epitope being immunodominant.Typically, antigenic peptides selected from the present proteins andpolypeptides will range from 5 to about 100 amino acids in length. Moretypically, however, such an antigenic peptide will be a maximum of about50 amino acids in length, and preferably a maximum of about 30 aminoacids. It is usually desirable to select a sequence of about 10, 12 or15 amino acids, up to about 20 or 25 amino acids.

Amino acid sequences comprising useful epitopes may be identified in anumber of ways. For example, preparing a series of short peptides thattaken together span the entire protein sequence may be used to screenthe entire protein sequence. One of skill in the art may routinely testa few large polypeptides for the presence of an epitope showing adesired reactivity and also test progressively smaller and overlappingfragments to identify a preferred epitope with the desired specificityand reactivity.

Antigenic polypeptides and peptides are useful for the production ofmonoclonal and polyclonal antibodies. Antibodies to a polypeptideencoded by the polynucleotides of NSEQ, polypeptide analogs or portionsthereof, may be generated using methods that are well known in the art.Such antibodies may include, but are not limited to, polyclonal,monoclonal, chimeric, and single chain antibodies, Fab fragments, andfragments produced by a Fab expression library. Neutralizing antibodies,such as those that inhibit dimer formation, are especially preferred fortherapeutic use. Monoclonal antibodies may be prepared using anytechnique that provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma, the human B-cell hybridoma, and the EBV-hybridomatechniques. In addition, techniques developed for the production ofchimeric antibodies may be used. Alternatively, techniques described forthe production of single chain antibodies may be employed. Fabs that maycontain specific binding sites for a polypeptide encoded by thepolynucleotides of NSEQ, or a portion thereof, may also be generated.Various immunoassays may be used to identify antibodies having thedesired specificity. Numerous protocols for competitive binding orimmunoradiometric assays using either polyclonal or monoclonalantibodies with established specificities are well known in the art.

To obtain polyclonal antibodies, a selected animal may be immunized witha protein or polypeptide. Serum from the animal may be collected andtreated according to known procedures. Polyclonal antibodies to theprotein or polypeptide of interest may then be purified by affinitychromatography. Techniques for producing polyclonal antisera are wellknown in the art.

Monoclonal antibodies (Mabs) may be made by one of several proceduresavailable to one of skill in the art, for example, by fusing antibodyproducing cells with immortalized cells and thereby making a hybridoma.The general methodology for fusion of antibody producing B cells to animmortal cell line is well within the province of one skilled in theart. Another example is the generation of Mabs from mRNA extracted frombone marrow and spleen cells of immunized animals using combinatorialantibody library technology.

The major drawback of Mabs derived from animals or from derived celllines is that although they may be administered to a patient fordiagnostic or therapeutic purposes, they are often recognized as foreignantigens by the immune system and are unsuitable for continued use.Antibodies that are not recognized as foreign antigens by the humanimmune system have greater potential for both diagnosis and treatment.Methods for generating human and humanized antibodies are now well knownin the art.

Chimeric antibodies may be constructed in which regions of a non-humanMab are replaced by their human counterparts. A preferred chimericantibody is one that has amino acid sequences that comprise one or morecomplementarity determining regions (CDRs) of a non-human Mab that bindsto a polypeptide encoded by the polynucleotides of NSEQ, or a portionthereof, grafted to human framework (FW) regions. Methods for producingsuch antibodies are well known in the art. Amino acid residuescorresponding to CDRs and FWs are known to one of average skill in theart.

A variety of methods have been developed to preserve or to enhanceaffinity for antigen of antibodies comprising grafted CDRs. One way isto include in the chimeric antibody the foreign framework residues thatinfluence the conformation of the CDR regions. A second way is to graftthe foreign CDRs onto human variable domains with the closest homologyto the foreign variable region. Thus, grafting of one or more non-humanCDRs onto a human antibody may also involve the substitution of aminoacid residues which are adjacent to a particular CDR sequence or whichare not contiguous with the CDR sequence but which are packed againstthe CDR in the overall antibody variable domain structure and whichaffect the conformation of the CDR. Humanized antibodies of theinvention therefore include human antibodies which comprise one or morenon-human CDRs as well as such antibodies in which additionalsubstitutions or replacements have been made to preserve or enhancebinding characteristics.

Chimeric antibodies of the invention also include antibodies that havebeen humanized by replacing surface-exposed residues to make the Mabappear human. Because the internal packing of amino acid residues in thevicinity of the antigen-binding site remains unchanged, affinity ispreserved. Substitution of surface-exposed residues of a polypeptideencoded by the polynucleotides of NSEQ (or a portion thereof)-antibodyaccording to the invention for the purpose of humanization does not meansubstitution of CDR residues or adjacent residues that influenceaffinity for a polypeptide encoded by the polynucleotides of NSEQ, or aportion thereof.

Chimeric antibodies may also include antibodies where some or allnon-human constant domains have been replaced with human counterparts.This approach has the advantage that the antigen-binding site remainsunaffected. However, significant amounts of non-human sequences may bepresent where variable domains are derived entirely from non-humanantibodies.

Antibodies of the invention include human antibodies that are antibodiesconsisting essentially of human sequences. Human antibodies may beobtained from phage display libraries wherein combinations of humanheavy and light chain variable domains are displayed on the surface offilamentous phage. Combinations of variable domains are typicallydisplayed on filamentous phage in the form of Fab's or scFvs. Thelibrary may be screened for phage bearing combinations of variabledomains having desired antigen-binding characteristics. Preferredvariable domain combinations are characterized by high affinity for apolypeptide encoded by the polynucleotides of NSEQ, or a portionthereof. Preferred variable domain combinations may also becharacterized by high specificity for a polypeptide encoded by thepolynucleotides of NSEQ, or a portion thereof, and littlecross-reactivity to other related antigens. By screening from very largerepertoires of antibody fragments, (2-10×10¹⁰) a good diversity of highaffinity Mabs may be isolated, with many expected to have sub-nanomolaraffinities for a polypeptide encoded by the polynucleotides of NSEQ, ora portion thereof.

Alternatively, human antibodies may be obtained from transgenic animalsinto which un-rearranged human Ig gene segments have been introduced andin which the endogenous mouse Ig genes have been inactivated. Preferredtransgenic animals contain very large contiguous Ig gene fragments thatare over 1 Mb in size but human polypeptide-specific Mabs of moderateaffinity may be raised from transgenic animals containing smaller geneloci. Transgenic animals capable of expressing only human Ig genes mayalso be used to raise polyclonal antiserum comprising antibodies solelyof human origin.

Antibodies of the invention may include those for which bindingcharacteristics have been improved by direct mutation or by methods ofaffinity maturation. Affinity and specificity may be modified orimproved by mutating CDRs and screening for antigen binding sites havingthe desired characteristics. CDRs may be mutated in a variety of ways.One way is to randomize individual residues or combinations of residuesso that in a population of otherwise identical antigen binding sites,all twenty amino acids may be found at particular positions.Alternatively, mutations may be induced over a range of CDR residues byerror prone PCR methods. Phage display vectors containing heavy andlight chain variable region gene may be propagated in mutator strains ofE. coli. These methods of mutagenesis are illustrative of the manymethods known to one of skill in the art.

Antibodies of the invention may include complete anti-polypeptideantibodies as well as antibody fragments and derivatives that comprise abinding site for a polypeptide encoded by the polynucleotides of NSEQ,or a portion thereof. Derivatives are macromolecules that comprise abinding site linked to a functional domain. Functional domains mayinclude, but are not limited to signalling domains, toxins, enzymes andcytokines.

The antibodies obtained by the means described herein may be useful fordetecting proteins, variant and derivative polypeptides in specifictissues or in body fluids. Moreover, detection of aberrantly expressedproteins or protein fragments is probative of a disease state. Forexample, expression of the present polypeptides encoded by thepolynucleotides of NSEQ, or a portion thereof, may indicate that theprotein is being expressed at an inappropriate rate or at aninappropriate developmental stage. Hence, the present antibodies may beuseful for detecting diseases associated with protein expression fromNSEQs disclosed herein.

A variety of protocols for measuring polypeptides, including ELISAs,RIAs, and FACS, are well known in the art and provide a basis fordiagnosing altered or abnormal levels of expression. Standard values forpolypeptide expression are established by combining samples taken fromhealthy subjects, preferably human, with antibody to the polypeptideunder conditions for complex formation. The amount of complex formationmay be quantified by various methods, such as photometric means.Quantities of polypeptide expressed in disease samples may be comparedwith standard values. Deviation between standard and subject values mayestablish the parameters for diagnosing or monitoring disease.

Design of immunoassays is subject to a great deal of variation and avariety of these are known in the art. Immunoassays may use a monoclonalor polyclonal antibody reagent that is directed against one epitope ofthe antigen being assayed. Alternatively, a combination of monoclonal orpolyclonal antibodies may be used which are directed against more thanone epitope. Protocols may be based, for example, upon competition whereone may use competitive drug screening assays in which neutralizingantibodies capable of binding a polypeptide encoded by thepolynucleotides of NSEQ, or a portion thereof, specifically compete witha test compound for binding the polypeptide. Alternatively one may use,direct antigen-antibody reactions or sandwich type assays and protocolsmay, for example, make use of solid supports or immunoprecipitation.Furthermore, antibodies may be labelled with a reporter molecule foreasy detection. Assays that amplify the signal from a bound reagent arealso known. Examples include immunoassays that utilize avidin andbiotin, or which utilize enzyme-labelled antibody or antigen conjugates,such as ELISA assays.

Kits suitable for immunodiagnosis and containing the appropriatelabelled reagents include antibodies directed against the polypeptideprotein epitopes or antigenic regions, packaged appropriately with theremaining reagents and materials required for the conduct of the assay,as well as a suitable set of assay instructions.

The present invention therefore provides a kit for specifically assayinga polypeptide described herein, the kit may comprise, for example, anantibody or antibody fragment capable of binding specifically to thepolypeptide described herein.

In accordance with the present invention, the kit may be a diagnostickit, which may comprise:

-   -   a) one or more antibodies described herein; and    -   b) a detection reagent which may comprise a reporter group.

In accordance with the present invention, the antibodies may beimmobilized on a solid support. The detection reagent may comprise, forexample, an anti-immunoglobulin, protein G, protein A or lectin etc. Thereporter group may be selected, without limitation, from the groupconsisting of radioisotopes, fluorescent groups, luminescent groups,enzymes, biotin and dye particles.

In yet another aspect of the invention, an NSEQ, a portion thereof, orits complement, may be used therapeutically for the purpose ofexpressing mRNA and polypeptide, or conversely to block transcription ortranslation of the mRNA. Expression vectors may be constructed usingelements from retroviruses, adenoviruses, herpes or vaccinia viruses, orbacterial plasmids, and the like. These vectors may be used for deliveryof nucleotide sequences to a particular target organ, tissue, or cellpopulation. Methods well known to those skilled in the art may be usedto construct vectors to express nucleic acid sequences or theircomplements.

Alternatively, NSEQ, a portion thereof, or its complement, may be usedfor somatic cell or stem cell gene therapy. Vectors may be introduced invivo, in vitro, and ex vivo. For ex vivo therapy, vectors are introducedinto stem cells taken from the subject, and the resulting transgeniccells are clonally propagated for autologous transplant back into thatsame subject. Delivery of NSEQ by transfection, liposome injections, orpolycationic amino polymers may be achieved using methods that are wellknown in the art. Additionally, endogenous NSEQ expression may beinactivated using homologous recombination methods that insert aninactive gene sequence into the coding region or other targeted regionof NSEQ.

Vectors containing NSEQ may be transformed into a cell or tissue toexpress a missing polypeptide or to replace a non-functionalpolypeptide. Similarly a vector constructed to express the complement ofNSEQ may be transformed into a cell to down-regulate the over-expressionof a polypeptide encoded by the polynucleotides of NSEQ, or a portionthereof. Complementary or anti-sense sequences may consist of anoligonucleotide derived from the transcription initiation site;nucleotides between about positions −10 and +10 from the ATG arepreferred. Similarly, inhibition may be achieved using triple helixbase-pairing methodology. Triple helix pairing is useful because itcauses inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature. (See, e.g., Gee et al. 1994)

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thecleavage of mRNA and decrease the levels of particular mRNAs, such asthose comprising the polynucleotide sequences of the invention.Ribozymes may cleave mRNA at specific cleavage sites. Alternatively,ribozymes may cleave mRNAs at locations dictated by flanking regionsthat form complementary base pairs with the target mRNA. Theconstruction and production of ribozymes is well known in the art.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends of the molecule,or the use of phosphorothioate or 2′ O-methyl rather than phosphodiesterlinkages within the backbone of the molecule. Alternatively,nontraditional bases such as inosine, queosine, and wybutosine, as wellas acetyl-, methyl-, thio-, and similarly modified forms of adenine,cytidine, guanine, thymine, and uridine which are not as easilyrecognized by endogenous endonucleases, may be included.

One of skill in the art will readily appreciate that antibodies andantibody conjugates of the invention, where used in the human body forthe purpose of the therapeutic applications discussed above, may beadministered in the form of a composition. Such pharmaceuticalcompositions may consist of a polypeptide encoded by the polynucleotidesof NSEQ, a portion thereof, or antibodies, mimetics, agonists,antagonists, or inhibitors of the polypeptide. The compositions may beadministered alone or in combination with at least one other agent, suchas a stabilizing compound, which may be administered in any sterile,biocompatible pharmaceutical carrier including, but not limited to,saline, buffered saline, dextrose, and water. The compositions may beadministered to a subject alone or in combination with other agents,drugs, or hormones.

In addition to the active ingredients, these pharmaceutical compositionsmay contain pharmaceutically acceptable carriers comprising excipientsand auxiliaries that facilitate processing of the active compounds intopreparations that may be used pharmaceutically. Further details ontechniques for formulation and administration may be found in the latestedition of Remington's Pharmaceutical Sciences (Maack Publishing Co.,Easton Pa.).

For any compound, the therapeutically effective dose may be estimatedinitially either in cell culture assays or in animal models such asmice, rats, rabbits, dogs, or pigs. An animal model may also be used todetermine the concentration range and route of administration. Suchinformation may then be used to determine useful doses and routes foradministration in humans. These techniques are well known to one skilledin the art and a therapeutically effective dose refers to that amount ofactive ingredient that ameliorates the symptoms or condition.Therapeutic efficacy and toxicity may be determined by standardpharmaceutical procedures in cell cultures or with experimental animals,such as by calculating and contrasting the ED₅₀ (the dosetherapeutically effective in 50% of the population) and LD₅₀ (the doselethal to 50% of the population) statistics. Any of the therapeuticcompositions described above may be applied to any subject in need ofsuch therapy, including, but not limited to, mammals such as dogs, cats,cows, horses, rabbits, monkeys, and most preferably, humans.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

The term “Treatment” for purposes of this disclosure refers to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) the targeted pathologiccondition or disorder. Those in need of treatment include those alreadywith the disorder as well as those prone to have the disorder or thosein whom the disorder is to be prevented.

Use of NSEQ in General Research

The invention finally provides products, compositions, processes andmethods that utilize an NSEQ, their open reading frame, or a polypeptideencoded by the polynucleotides of NSEQ or their open reading frame, or aportion thereof, their variants, analogs and derivatives for research,biological, clinical and therapeutic purposes. For example, to identifysplice variants, mutations, and polymorphisms

NSEQ may be extended utilizing a partial nucleotide sequence andemploying various PCR-based methods known in the art to detect upstreamsequences such as promoters and other regulatory elements. Additionally,one may use an XL-PCR kit (PE Biosystems, Foster City Calif.), nestedprimers, and commercially available cDNA libraries (Life Technologies,Rockville Md.) or genomic libraries (Clontech, Palo Alto Calif.) toextend the sequence.

The polynucleotides may also be used as targets in a micro-array. Themicro-array may be used to monitor the expression patterns of largenumbers of genes simultaneously and to identify splice variants,mutations, and polymorphisms. Information derived from analyses of theexpression patterns may be used to determine gene function, tounderstand the genetic basis of a disease, to diagnose a disease, and todevelop and monitor the activities of therapeutic agents used to treat adisease. Microarrays may also be used to detect genetic diversity,single nucleotide polymorphisms which may characterize a particularpopulation, at the genomic level.

In yet another embodiment, polynucleotides may be used to generatehybridization probes useful in mapping the naturally occurring genomicsequence. Fluorescent in situ hybridization (FISH) may be correlatedwith other physical chromosome mapping techniques and genetic map data.

The present invention more particularly relates in one aspect thereof toa method of representatively identifying an endogeneously differentiallyexpressed sequence involved in osteoclast differentiation. The sequencemay be, for example, differentially expressed in a differentiatedosteoclast cell compared to an undifferentiated osteoclast precursorcell.

The method of the present invention may comprise;

-   -   a) separately providing total messenger RNA from differentiated        osteoclast cell and undifferentiated osteoclast precursor cell,        the total messenger RNA may comprise, for example, at least one        endogeneously differentially expressed sequence,    -   b) generating (e.g., single copy of a) single-stranded cDNA from        each messenger RNA of differentiated osteoclast cell and (e.g.,        randomly) tagging the 3′-end of the single-stranded cDNA with a        RNA polymerase promoter sequence and a first sequence tag;    -   c) generating (e.g., single copy of a) single-stranded cDNA from        each messenger RNA of undifferentiated osteoclast precursor cell        and (e.g., randomly) tagging the 3′-end of the single-stranded        cDNA with a RNA polymerase promoter sequence and a second        sequence tag;    -   d) separately generating partially or completely double-stranded        5′-tagged-DNA from each of b) and c), the double-stranded        5′-tagged-DNA may thus comprise in a 5′ to 3′ direction, a        double-stranded RNA polymerase promoter, a first or second        sequence tag and an endogenously expressed sequence,    -   e) separately linearly amplifying a first and second tagged        sense RNA from each of d) with a RNA polymerase enzyme (which        may be selected based on the promoter used for tagging),    -   f) generating single-stranded complementary first or second        tagged DNA from one of e),    -   g) hybridizing the single-stranded complementary first or second        tagged DNA of f) with the other linearly amplified sense RNA of        e),    -   h) recovering unhybridized RNA with the help of the first or        second sequence tag (for example by PCR or hybridization), and;    -   i) identifying (determining) the nucleotide sequence of        unhybridized RNA.

The method may further comprise the step of comparatively determiningthe presence of the identified endogeneously and differentiallyexpressed sequence in a differentiated osteoclast cell relative to anundifferentiated osteoclast precursor cell.

A sequence which is substantially absent (e.g., totally absent orpresent in very low quantity) from one of differentiated osteoclast cellor an undifferentiated osteoclast precursor cell and present in theother of differentiated osteoclast cell or an undifferentiatedosteoclast precursor cell may thus be selected.

In accordance with the present invention, the sequence may be furtherselected based on a reduced or substantially absent expression in othernormal tissue, therefore representing a candidate sequence specificallyinvolved in osteoclast differentiation and bone remodeling.

The method may also further comprise a step of determining the completesequence of the nucleotide sequence and may also comprise determiningthe coding sequence of the nucleotide sequence.

The present invention also relates in a further aspect, to the isolatedendogeneously and differentially expressed sequence (polynucleotide andpolypeptide) identified by the method of the present invention.

More particularly, the present invention encompasses a polynucleotidewhich may comprise the identified polynucleotide sequence, apolynucleotide which may comprise the open reading frame of theidentified polynucleotide sequence, a polynucleotide which may comprisea nucleotide sequence substantially identical to the polynucleotideidentified by the method of the present invention, a polynucleotidewhich may comprise a nucleotide sequence substantially complementary tothe polynucleotide identified by the method of the present invention,fragments and splice variant thereof, provided that the sequence doesnot consist in or comprise SEQ ID NO.:57.

In accordance with the present invention, the isolated endogeneously anddifferentially expressed sequence of the present invention may be acomplete or partial RNA molecule.

Isolated DNA molecule able to be transcribed into the RNA molecule ofthe present invention are also encompassed herewith as well as vectors(including expression vectors) comprising the such DNA or RNA molecule.

The present invention also relates to libraries comprising at least oneisolated endogeneously and differentially expressed sequence identifiedherein (e.g., partial or complete RNA or DNA, substantially identicalsequences or substantially complementary sequences (e.g., probes) andfragments thereof (e.g., oligonucleotides)).

In accordance with the present invention, the isolated endogeneously anddifferentially expressed sequence may be selected, for example, from thegroup consisting of a polynucleotide which may consist in or comprise;

-   -   a) any one of SEQ ID NO.:1 to SEQ ID NO.56, SEQ ID NO.: 83, SEQ        ID NO.:84 or SEQ ID NO.:87,    -   b) the open reading frame of any one of SEQ ID NO.:1 to SEQ ID        NO.56, SEQ ID NO.: 83, SEQ ID NO.:84 or SEQ ID NO.:87,    -   c) a polynucleotide which may comprise a nucleotide sequence        substantially identical to a) or b), and;    -   d) fragments of any one of a) to c).

Exemplary substantially identical sequence of a) or b) may comprise, forexample, a sequence which may be selected from the group consisting ofSEQ ID NO.:84, SEQ ID NO.:85, SEQ ID NO.:88, SEQ ID NO.:89 and the openreading frame of the SEQ ID NO.:84, SEQ ID NO.:85, SEQ ID NO.:88, SEQ IDNO.:89.

In a further aspect the present invention relates to a polypeptide whichmay be encoded by the isolated endogeneously and differentiallyexpressed sequence of the present invention.

Exemplary polypeptides may comprise a sequence selected from the groupconsisting of any one of SEQ ID NO.: 93 to 99, 101 to 155.

In accordance with the present invention, when the sequence is from anon-human mammal, the method further comprises identifying acorresponding human ortholog polynucleotide sequence using a methoddescribed herein or other methods known in the art.

The present invention therefore also relates to an isolated humanortholog polynucleotide sequence (involved in bone remodeling), the openreading frame of the human ortholog, substantially identical sequences,substantially complementary sequences, fragments and splice variantsthereof.

The present invention as well relates to an isolated polypeptide encodedby the human ortholog polynucleotide as well as biologically activeanalogs and biologically active fragments thereof.

Exemplary embodiments of human ortholog polynucleotides encompassedherewith include, for example, a sequence selected form the groupconsisting of SEQ ID NO.:84, SEQ ID NO.:85, SEQ ID NO.:88, SEQ ID NO.:89and the open reading frame of the SEQ ID NO.:84, SEQ ID NO.:85, SEQ IDNO.:88, SEQ ID NO.:89.

Exemplary embodiments of isolated polypeptide encoded by some humanorthologs identified herein include for example, a polypeptide selectedfrom the group consisting of SEQ ID NO.:150, SEQ ID NO.:153, SEQ IDNO.:154 and SEQ ID NO.:155.

The present invention also more particularly relates, in an additionalaspect thereof, to an isolated polynucleotide which may bedifferentially expressed in differentiated osteoclast cell compared toundifferentiated osteoclast precursor cell.

The isolated polynucleotide may comprise a member selected from thegroup consisting of;

-   -   a) a polynucleotide which may comprise any one of SEQ ID NO.:1        to SEQ ID NO.56, SEQ ID NO.: 83, SEQ ID NO.:86 or SEQ ID NO.:87,    -   b) a polynucleotide which may comprise the open reading frame of        any one of SEQ ID NO.:1 to SEQ ID NO.56, SEQ ID NO.: 83, SEQ ID        NO.:86 or SEQ ID NO.:87,    -   c) a polynucleotide which may comprise a sequence substantially        identical (e.g., from about 50 to 100%, or about 60 to 100% or        about 70 to 100% or about 80 to 100% or about 85, 90, 95 to 100%        identical over the entire sequence or portion of sequences)        to a) or b),    -   d) a polynucleotide which may comprise a sequence substantially        complementary (e.g., from about 50 to 100%, or about 60 to 100%        or about 70 to 100% or about 80 to 100% or about 85, 90, 95 to        100% complementarity over the entire sequence or portion of        sequences) to a) or b), and;    -   e) a fragment of any one of a) to d)    -   f) including polynucleotides which consist in the above.

Exemplary polynucleotides which are substantially identical to thoselisted above, includes for example, polynucleotides selected from thegroup consisting of SEQ ID NO.:84, SEQ ID NO.:85, SEQ ID NO.:88, SEQ IDNO.:89 and the open reading frame of any one of SEQ ID NO.:84, SEQ IDNO.:85, SEQ ID NO.:88 or SEQ ID NO.:89.

Exemplary polynucleotides fragments of those listed above comprisespolynucleotides of at least 10 nucleic acids which may be substantiallycomplementary to the nucleic acid sequence of any one of SEQ ID NO.: 1to 56 or SEQ ID NO.: 83 to SEQ ID NO.:89, such as, for example,fragments selected from the group consisting of any one of SEQ ID NO.:64 to 80 or 90.

The present invention also relates to an isolated polynucleotideinvolved in osteoclast differentiation, the isolated polynucleotide maybe selected, for example, from the group consisting of;

-   -   a) a polynucleotide comprising any one of SEQ ID NO.: 1 to 56 or        83 to 89,    -   b) a polynucleotide comprising the open reading frame of any one        of SEQ

ID NO.: 1 to 56 or 83 to 89, and;

-   -   c) a polynucleotide substantially identical to a) or b).

The present invention also further relates to an isolated polynucleotidewhich may be able to promote osteoclast differentiation (e.g., in amammal or mammalian cell thereof). The polynucleotide may be selected,for example, from the group consisting of polynucleotides which maycomprise;

-   -   a) any one of SEQ ID NO.:1 to 5, 8 to 56 or 83 to 89;    -   b) the open reading frame of any one of SEQ ID NO.:1 to 5, 8 to        56 or 83 to 89, and;    -   c) a sequence of at least 10 nucleic acids which may be        complementary to the nucleic acid sequence of any one of SEQ ID        NO.:6 or SEQ ID NO.:7.

In yet a further aspect, the present invention relates to an isolatedpolynucleotide which may be able to inhibit osteoclast differentiation(e.g., in a mammal or mammalian cell thereof). The polynucleotide may beselected, for example, from the group consisting of polynucleotideswhich may comprise;

-   -   a) any one of SEQ ID NO.:6 or SEQ ID NO.:7,    -   b) the open reading frame of any one of SEQ ID NO.:6 or SEQ ID        NO.:7, and;    -   c) a sequence of at least 10 nucleic acids which is        complementary to the nucleic acid sequence of any one of SEQ ID        NO.:1 to 5 or 8 to 57 or 83 to 89.

Suitable polynucleotides include, for example, a polynucleotide havingor comprising those which are selected from the group consisting of SEQID NO. 64 to 82 and 90.

Suitable polynucleotides may be those which may be able to inhibitosteoclast differentiation which has been induced by an inducer ofosteoclast differentiation such as those listed herein.

In accordance with the present invention, the polynucleotide may be, forexample, a RNA molecule, a DNA molecule, including those which arepartial or complete, single-stranded or double-stranded, hybrids, etc.

The present invention also relates to a vector (e.g., an expressionvector) comprising the polynucleotide of the present invention.

The present invention additionally relates in an aspect thereof to alibrary of polynucleotide sequences which may be differentiallyexpressed in a differentiated osteoclast cell compared to anundifferentiated osteoclast precursor cell. The library may comprise,for example, at least one member selected from the group consisting of

-   -   a) a polynucleotide which may comprise any one of SEQ ID NO.:1        to SEQ ID NO.57 and 83 to 89,    -   b) a polynucleotide which may comprise the open reading frame of        any one of SEQ ID NO.:1 to SEQ ID NO.57 and 83 to 89,    -   c) a polynucleotide which may comprise a sequence substantially        identical (e.g., from about 50 to 100%, or about 60 to 100% or        about 70 to 100% or about 80 to 100% or about 85, 90, 95 to 100%        identical over the entire sequence or portion of sequences)        to a) or b),    -   d) a polynucleotide which may comprise a sequence substantially        complementary (e.g., from about 50 to 100%, or about 60 to 100%        or about 70 to 100% or about 80 to 100% or about 85, 90, 95 to        100% complementarity over the entire sequence or portion of        sequences) to a) or b), and;    -   e) a fragment of any one of a) to d).

The present invention also relates to an expression library which maycomprise a library of polynucleotides described herein. In accordancewith the present invention, each of the polynucleotide may be containedwithin an expression vector.

Arrays and kits comprising a library of polynucleotide sequences(comprising at least one polynucleotide including complementarysequences) of the present invention are also encompassed herewith.

The present invention also provides in an additional aspect, apharmaceutical composition for inhibiting osteoclast differentiation(bone resorption and bone resorption related diseases or disorders), thepharmaceutical composition may comprise, for example;

-   -   a) an isolated polynucleotide as defined herein (e.g., able to        inhibit osteoclast differentiation) and;    -   b) a pharmaceutically acceptable carrier.

The present invention also provides in yet an additional aspect, amethod for inhibiting osteoclast differentiation (e.g., for inhibitingbone resorption or for ameliorating bone resorption) in a mammal(individual) in need thereof (or in a mammalian cell), the method maycomprise administering an isolated polynucleotide (e.g., able to inhibitosteoclast differentiation) or a suitable pharmaceutical composition.

In accordance with the present invention, the mammal in need may suffer,for example and without limitation, from a condition selected from thegroup consisting of osteoporosis, osteopenia, osteomalacia,hyperparathyroidism, hyperthyroidism, hypogonadism, thyrotoxicosis,systemic mastocytosis, adult hypophosphatasia, hyperadrenocorticism,osteogenesis imperfecta, Paget's disease, Cushing's disease/syndrome,Turner syndrome, Gaucher disease, Ehlers-Danlos syndrome, Marfan'ssyndrome, Menkes' syndrome, Fanconi's syndrome, multiple myeloma,hypercalcemia, hypocalcemia, arthritides, periodontal disease, rickets(including vitamin D dependent, type I and II, and x-linkedhypophosphatemic rickets), fibrogenesis imperfecta ossium,osteosclerotic disorders such as pycnodysostosis and damage caused bymacrophage-mediated inflammatory processes, etc.

In a further aspect, the present invention relates to the use of anisolated polynucleotide (e.g., able to inhibit osteoclastdifferentiation) for the preparation of a medicament for the treatmentof a bone resorption disease.

The present invention in another aspect thereof, provides apharmaceutical composition for promoting osteoclast differentiation in amammal in need thereof. The pharmaceutical composition may comprise, forexample;

-   -   a. an isolated polynucleotide (e.g., able to promote osteoclast        differentiation) and;    -   b. a pharmaceutically acceptable carrier.

The present invention also further provides a method for promotingosteoclast differentiation in a mammal in need thereof (or in amammalian cell), the method may comprise, for example, administering anisolated polynucleotide (e.g., able to promote osteoclastdifferentiation) or a suitable pharmaceutical composition as describedabove.

The present invention additionally relates to the use of an isolatedpolynucleotide (e.g., able to promote osteoclast differentiation) forthe preparation of a medicament for the treatment of a diseaseassociated with insufficient bone resorption (e.g., hyperostosis).

The present invention also relates to the use of at least onepolynucleotide which may be selected from the group consisting of;

-   -   a) a polynucleotide comprising the any one of SEQ ID NO.:1 to        SEQ ID NO.57 and 83 to 89,    -   b) a polynucleotide comprising the open reading frame of any one        of SEQ ID NO.:1 to SEQ ID NO.57 and 83 to 89,    -   c) a polynucleotide comprising a sequence substantially        identical (e.g., from about 50 to 100%, or about 60 to 100% or        about 70 to 100% or about 80 to 100% or about 85, 90, 95 to 100%        identical over the entire sequence or portion of sequences)        to a) or b),    -   d) a polynucleotide comprising a sequence substantially        complementary (e.g., from about 50 to 100%, or about 60 to 100%        or about 70 to 100% or about 80 to 100% or about 85, 90, 95 to        100% complementarity over the entire sequence or portion of        sequences) to a) or b),    -   e) a fragment of any one of a) to d) and;    -   f) a library comprising any one of a) to d) in the diagnosis of        a condition related to bone remodeling.

Also encompassed by the present invention are kits for the diagnosis ofa condition related to bone remodeling. The kit may comprise, forexample, at least one sequence substantially complementary to any one ofSEQ ID NO.:1 to SEQ ID NO.57 or 83 to 89, the open reading frame of anyone of SEQ ID NO.:1 to SEQ ID NO.57 or 83 to 89 and fragments thereof.

The present invention also provides in an additional aspect, an isolatedpolypeptide (polypeptide sequence) which may be able to promoteosteoclast differentiation (in a mammal or a mammalian cell thereof).The polypeptide may comprise (or consist in) a sequence selected fromthe group consisting of;

-   -   a) any one of SEQ ID NO.: 93 to 97 or 101 to 155,    -   b) a biologically active fragment of any one of a),    -   c) a biologically active analog of any one of a).

In accordance with the present invention, the biologically active analogmay comprise, for example, at least one conservative amino acidsubstitution compared to the original sequence.

In yet a further aspect, the present invention provides a pharmaceuticalcomposition for promoting osteoclast differentiation (e.g., forpromoting bone resorption). The pharmaceutical composition may comprise,for example a polypeptide (e.g., able to promote osteoclastdifferentiation) and a pharmaceutically acceptable carrier.

Methods for promoting osteoclast differentiation in a mammal in needthereof (or in a mammalian cell) are also provided by the presentinvention, which methods may comprise administering an isolatedpolypeptide (e.g., able to promote osteoclast differentiation) orsuitable pharmaceutical composition described herein.

In additional aspects, the present invention relates to the use of anisolated polypeptide (e.g., able to promote osteoclast differentiation)for the preparation of a medicament for the treatment of a diseaseassociated with insufficient bone resorption.

In a further aspect, the present invention relates to an isolatedpolypeptide able to inhibit osteoclast differentiation (in a mammal ormammalian cell thereof), the polypeptide may comprise, for example, asequence selected from the group consisting of

-   -   a) a sequence which may comprise or consist in any one of SEQ ID        NO.:98 and SEQ ID NO.:99,    -   b) a biologically active fragment of any one of a),    -   c) a biologically active analog of any one of a).

In accordance with the present invention, the biologically active analogmay comprise, for example, at least one conservative amino acidsubstitution in the amino acid sequence in comparison to anotherpolypeptide

The present invention further encompasses pharmaceutical compositionswhich may comprise the isolated polypeptide described herein.

Methods for ameliorating bone resorption in an individual in needthereof are also encompassed herewith, which method may comprise, forexample, administering an isolated polypeptide (e.g., able to inhibitosteoclast differentiation) or suitable pharmaceutical compositionswhich may comprise such polypeptide.

In a further aspect the present invention provides a method forameliorating bone resorption in an individual in need thereof which maycomprise administering a compound capable of inhibiting (e.g., in anosteoclast precursor cell) the activity or expression of a polypeptideinvolved in (or able to promote) osteoclast differentiation such as forexample, a polypeptide selected from the group consisting of SEQ ID NO.:93 to 97 and 101 to 155.

In accordance with the present invention, the mammal may suffer, forexample, from a condition selected from the group consisting ofosteoporosis, osteopenia, osteomalacia, hyperparathyroidism,hyperthyroidism, hypogonadism, thyrotoxicosis, systemic mastocytosis,adult hypophosphatasia, hyperadrenocorticism, osteogenesis imperfecta,Paget's disease, Cushing's disease/syndrome, Turner syndrome, Gaucherdisease, Ehlers-Danlos syndrome, Marfan's syndrome, Menkes' syndrome,Fanconi's syndrome, multiple myeloma, hypercalcemia, hypocalcemia,arthritides, periodontal disease, rickets (including vitamin Ddependent, type I and II, and x-linked hypophosphatemic rickets),fibrogenesis imperfecta ossium, osteosclerotic disorders such aspycnodysostosis and damage caused by macrophage-mediated inflammatoryprocesses, etc.

In yet a further aspect, the present invention relates to the use of apolypeptide able to inhibit osteoclast differentiation in thepreparation of a medicament for the treatment of a bone resorptiondisease in an individual in need thereof.

The present invention also relates to the use of a compound able toinhibit (e.g., in an osteoclast precursor cell) the activity orexpression of a polypeptide which may be selected, for example, from thegroup consisting of SEQ ID NO.: 93 to 97 and 101 to 155 in thepreparation of a medicament for the treatment of a bone resorptiondisease in an individual in need thereof.

Antibodies and antigen-binding fragment thereof which are able to bindto any of the polypeptide described herein, including those which may beselected from the group consisting of SEQ ID NO.: 93 to 97 and 101 to155 are also encompassed by the present invention.

In accordance with the present invention, the antibody may be able, forexample, to inhibit osteoclast differentiation.

The present invention also relates to a composition (e.g.,pharmaceutical composition) which may comprise;

-   -   a) the antibody of claim 40 and;    -   b) a pharmaceutically acceptable carrier.

The present invention relates in a further aspect to a method ofinhibiting osteoclast differentiation which may comprise administeringto a mammal in need thereof the antibody described herein or apharmaceutical composition comprising such antibody.

The present invention relates in yet a further aspect to the use of anantibody as defined herein for the preparation of a medicament for thetreatment of a bone resorption disease in an individual in need thereof.

In an additional aspect, the present invention relates to an immunizingcomposition which may comprise a polypeptide, such as a polypeptideselected from the group consisting of SEQ ID NO.: 93 to 155, analogs orfragments thereof or a nucleic acid (polynucleotide) selected, forexample, from the group consisting of those comprising or consisting in(a) SEQ ID NO.: 1 to 56 and 83 to 89, (b) a polynucleotide which maycomprise the open reading frame of SEQ ID NO.: 1 to 56 and 83 to 89, (c)substantially identical sequences of any one of (a) or (b) or fragmentsof any one of (a), (b) or (c) able to encode immunologically activepolypeptides thereof.

In yet an additional aspect, the present invention relates to a methodof diagnosing a condition related to a bone resorption disorder ordisease in an individual in need thereof. The method may comprise, forexample, quantifying a polynucleotide described herein, such as, forexample, those selected from the group consisting of those comprising orconsisting of (a) SEQ ID NO.:1 to 56 and 83 to 89 (b) a polynucleotidewhich may comprise the open reading frame of SEQ ID NO.: 1 to 56 and 83to 89, (c) substantially identical sequences of any one of (a) or (b),or a polypeptide sequence which may be selected, for example, from thegroup consisting of 93 to 155 and analogs thereof in a sample from theindividual compared to a standard or normal value.

In an additional aspect, the present invention provides a method foridentifying an inhibitory compound (inhibitor, antagonist) which may beable to impair the function (activity) or expression of a polypeptidedescribed herein, such as, for example, those which may be selected fromthe group consisting of SEQ ID NO.: 93 to 97 and 100 to 155 and analogsthereof. The method may comprise contacting the polypeptide or a cellexpressing the polypeptide with a candidate compound and measuring thefunction (activity) or expression of the polypeptide. A reduction in thefunction or activity of the polypeptide (compared to the absence of thecandidate compound) may positively identify a suitable inhibitorycompound.

In accordance with the present invention, the impaired function oractivity may be associated with a reduced ability of the polypeptide topromote osteoclast differentiation, such as osteoclast differentiationinduced by an inducer described herein or known in the art.

In accordance with the present invention the cell may not naturally(endogenously) express (polypeptide may substantially be unexpressed ina cell) the polypeptide or analog or alternatively, the expression of anaturally expressed polypeptide analog may be repressed.

For example, suitable method of screening for an inhibitor of SEQ IDNO.:153, may comprise repressing the expression of SEQ ID NO.:93 in amouse osteoclast cell and evaluating differentiation of the osteoclastcell in the presence or absence of a candidate inhibitor.

The impaired function or activity may also be associated with a reducedability of the polypeptide to interact with a known partner.

For example, suitable method of screening for an inhibitor of SEQ IDNO.: 154 may comprise measuring (evaluating) the interaction of thepolypeptide with the v-ATPase-a3 subunit in the presence or absence of acandidate inhibitor.

The present invention also provides a method for identifying aninhibitory compound (inhibitor, antagonist) able to impair the function(activity) or expression of a polypeptide such as, for example SEQ IDNO.: 98 or SEQ ID NO.:99. The method may comprise, for example,contacting the polypeptide or a cell expressing the polypeptide with acandidate compound and measuring the function (activity) or expressionof the polypeptide. A reduction in the function or activity of thepolypeptide (compared to the absence of the candidate compound) may thuspositively identify a suitable inhibitory compound.

In accordance with the present invention, the impaired function oractivity may be associated, for example, with a reduced ability of thepolypeptide to inhibit osteoclast differentiation.

The cell used to carry the screening test may not naturally(endogenously) express the polypeptide or analogs, or alternatively theexpression of a naturally expressed polypeptide analog may be repressed.

As used herein the term “sequence identity” relates to (consecutive)nucleotides of a nucleotide sequence which with reference to an originalnucleotide sequence. The identity may be compared over a region or overthe total sequence of a nucleic acid sequence.

Thus, “identity” may be compared, for example, over a region of 3, 4, 5,10, 19, 20 nucleotides or more (and any number there between). It is tobe understood herein that gaps of non-identical nucleotides may be foundbetween identical nucleic acids. For example, a polynucleotide may have100% identity with another polynucleotide over a portion thereof.However, when the entire sequence of both polynucleotide is compared,the two polynucleotides may have 50% of their overall (total) sequenceidentical to one another.

Polynucleotides of the present invention or portion thereof having fromabout 50 to about 100%, or about 60 to about 100% or about 70 to about100% or about 80 to about 100% or about 85%, about 90%, about 95% toabout 100% sequence identity with an original polynucleotide areencompassed herewith. It is known by those of skill in the art, that apolynucleotide having from about 50% to 100% identity may function(e.g., anneal to a substantially complementary sequence) in a mannersimilar to an original polynucleotide and therefore may be used inreplacement of an original polynucleotide. For example a polynucleotide(a nucleic acid sequence) may comprise or have from about 50% to 100%identity with an original polynucleotide over a defined region and maystill work as efficiently or sufficiently to achieve the presentinvention.

Percent identity may be determined, for example, with n algorithm GAP,BESTFIT, or FASTA in the Wisconsin Genetics Software Package Release7.0, using default gap weights.

As used herein the terms “sequence complementarity” refers to(consecutive) nucleotides of a nucleotide sequence which arecomplementary to a reference (original) nucleotide sequence. Thecomplementarity may be compared over a region or over the total sequenceof a nucleic acid sequence.

Polynucleotides of the present invention or portion thereof having fromabout 50 to about 100%, or about 60 to about 100% or about 70 to about100% or about 80 to about 100% or about 85%, about 90%, about 95% toabout 100% sequence complementarity with an original polynucleotide arethus encompassed herewith. It is known by those of skill in the art,that an polynucleotide having from about 50% to 100% complementaritywith an original sequence may anneal to that sequence in a mannersufficient to carry out the present invention (e.g., inhibit expressionof the original polynucleotide).

An “analogue” is to be understood herein as a molecule having abiological activity and chemical structure similar to that of apolypeptide described herein. An “analogue” may have sequence similaritywith that of an original sequence or a portion of an original sequenceand may also have a modification of its structure as discussed herein.For example, an “analogue” may have at least 90% sequence similaritywith an original sequence or a portion of an original sequence. An“analogue” may also have, for example; at least 70% or even 50% sequencesimilarity (or less, i.e., at least 40%) with an original sequence or aportion of an original sequence.

Also, an “analogue” may have, for example, at least 50% sequencesimilarity to an original sequence with a combination of one or moremodification in a backbone or side-chain of an amino acid, or anaddition of a group or another molecule, etc.

“Polynucleotide” generally refers to any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA, or modifiedRNA or DNA. “Polynucleotides” include, without limitation single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis a mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term polynucleotide also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications may be made to DNA and RNA; thus“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” includes but is not limited to linear andend-closed molecules. “Polynucleotide” also embraces relatively shortpolynucleotides, often referred to as oligonucleotides.

“Polypeptides” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds (i.e., peptide isosteres). “Polypeptide” refers to both shortchains, commonly referred as peptides, oligopeptides or oligomers, andto longer chains generally referred to as proteins. As described above,polypeptides may contain amino acids other than the 20 gene-encodedamino acids.

As used herein the term “polypeptide analog” relates to mutants,variants, chimeras, fusions, deletions, additions and any other type ofmodifications made relative to a given polypeptide.

As used herein the term “biologically active” refers to a variant orfragment which retains some or all of the biologicval activity of thenatural polypeptide, i.e., to be able to promote or inhibit osteoclastdifferentiation.

Thus, biologically active polypeptides in the form of the originalpolypeptides, fragments (modified or not), analogues (modified or not),derivatives (modified or not), homologues, (modified or not) of thepolypeptides described herein are encompassed by the present invention.

Therefore, any polypeptide having a modification compared to an originalpolypeptide which does not destroy significantly a desired biologicalactivity is encompassed herein. It is well known in the art, that anumber of modifications may be made to the polypeptides of the presentinvention without deleteriously affecting their biological activity.These modifications may, on the other hand, keep or increase thebiological activity of the original polypeptide or may optimize one ormore of the particularity (e.g. stability, bioavailability, etc.) of thepolypeptides of the present invention which, in some instance might bedesirable. Polypeptides of the present invention may comprise forexample, those containing amino acid sequences modified either bynatural processes, such as posttranslational processing, or by chemicalmodification techniques which are known in the art. Modifications mayoccur anywhere in a polypeptide including the polypeptide backbone, theamino acid side-chains and the amino- or carboxy-terminus. It will beappreciated that the same type of modification may be present in thesame or varying degrees at several sites in a given polypeptide. Also, agiven polypeptide may contain many types of modifications. It is to beunderstood herein that more than one modification to the polypeptidesdescribed herein are encompassed by the present invention to the extentthat the biological activity is similar to the original (parent)polypeptide.

As discussed above, polypeptide modification may comprise, for example,amino acid insertion (i.e., addition), deletion and substitution (i.e.,replacement), either conservative or non-conservative (e.g., D-aminoacids, desamino acids) in the polypeptide sequence where such changes donot substantially alter the overall biological activity of thepolypeptide.

Example of substitutions may be those, which are conservative (i.e.,wherein a residue is replaced by another of the same general type orgroup) or when wanted, non-conservative (i.e., wherein a residue isreplaced by an amino acid of another type). In addition, a non-naturallyoccurring amino acid may substitute for a naturally occurring amino acid(i.e., non-naturally occurring conservative amino acid substitution or anon-naturally occurring non-conservative amino acid substitution).

As is understood, naturally occurring amino acids may be sub-classifiedas acidic, basic, neutral and polar, or neutral and non-polar.Furthermore, three of the encoded amino acids are aromatic. It may be ofuse that encoded polypeptides differing from the determined polypeptideof the present invention contain substituted codons for amino acids,which are from the same type or group as that of the amino acid to bereplaced. Thus, in some cases, the basic amino acids Lys, Arg and Hismay be interchangeable; the acidic amino acids Asp and Glu may beinterchangeable; the neutral polar amino acids Ser, Thr, Cys, Gln, andAsn may be interchangeable; the non-polar aliphatic amino acids Gly,Ala, Val, Ile, and Leu are interchangeable but because of size Gly andAla are more closely related and Val, Ile and Leu are more closelyrelated to each other, and the aromatic amino acids Phe, Trp and Tyr maybe interchangeable.

It should be further noted that if the polypeptides are madesynthetically, substitutions by amino acids, which are not naturallyencoded by DNA (non-naturally occurring or unnatural amino acid) mayalso be made.

A non-naturally occurring amino acid is to be understood herein as anamino acid which is not naturally produced or found in a mammal. Anon-naturally occurring amino acid comprises a D-amino acid, an aminoacid having an acetylaminomethyl group attached to a sulfur atom of acysteine, a pegylated amino acid, etc. The inclusion of a non-naturallyoccurring amino acid in a defined polypeptide sequence will thereforegenerate a derivative of the original polypeptide. Non-naturallyoccurring amino acids (residues) include also the omega amino acids ofthe formula NH₂(CH₂)_(n)COOH wherein n is 2-6, neutral nonpolar aminoacids, such as sarcosine, t-butyl alanine, t-butyl glycine, N-methylisoleucine, norleucine, etc. Phenylglycine may substitute for Trp, Tyror Phe; citrulline and methionine sulfoxide are neutral nonpolar,cysteic acid is acidic, and ornithine is basic. Proline may besubstituted with hydroxyproline and retain the conformation conferringproperties.

It is known in the art that analogues may be generated by substitutionalmutagenesis and retain the biological activity of the polypeptides ofthe present invention. These analogues have at least one amino acidresidue in the protein molecule removed and a different residue insertedin its place. For example, one site of interest for substitutionalmutagenesis may include but are not restricted to sites identified asthe active site(s), or immunological site(s). Other sites of interestmay be those, for example, in which particular residues obtained fromvarious species are identical. These positions may be important forbiological activity. Examples of substitutions identified as“conservative substitutions” are shown in Table A. If such substitutionsresult in a change not desired, then other type of substitutions,denominated “exemplary substitutions” in Table A, or as furtherdescribed herein in reference to amino acid classes, are introduced andthe products screened.

In some cases it may be of interest to modify the biological activity ofa polypeptide by amino acid substitution, insertion, or deletion. Forexample, modification of a polypeptide may result in an increase in thepolypeptide's biological activity, may modulate its toxicity, may resultin changes in bioavailability or in stability, or may modulate itsimmunological activity or immunological identity. Substantialmodifications in function or immunological identity are accomplished byselecting substitutions that differ significantly in their effect onmaintaining (a) the structure of the polypeptide backbone in the area ofthe substitution, for example, as a sheet or helical conformation. (b)the charge or hydrophobicity of the molecule at the target site, or (c)the bulk of the side chain. Naturally occurring residues are dividedinto groups based on common side chain properties:

-   -   (1) hydrophobic: norleucine, methionine (Met), Alanine (Ala),        Valine (Val), Leucine (Leu), Isoleucine (Ile)    -   (2) neutral hydrophilic: Cysteine (Cys), Serine (Ser), Threonine        (Thr)    -   (3) acidic: Aspartic acid (Asp), Glutamic acid (Glu)    -   (4) basic: Asparagine (Asn), Glutamine (Gin), Histidine (His),        Lysine (Lys), Arginine (Arg)    -   (5) residues that influence chain orientation: Glycine (Gly),        Proline (Pro); and aromatic: Tryptophan (Trp), Tyrosine (Tyr),        Phenylalanine (Phe)

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another.

TABLE A Examplary amino acid substitution Conservative Original residueExemplary substitution substitution Ala (A) Val, Leu, Ile Val Arg (R)Lys, Gln, Asn Lys Asn (N) Gln, His, Lys, Arg Gln Asp (D) Glu Glu Cys (C)Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro Pro His (H) Asn,Gln, Lys, Arg Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu norleucine Leu(L) Norleucine, Ile, Val, Met, Ile Ala, Phe Lys (K) Arg, Gln, Asn ArgMet (M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala Leu Pro (P) Gly GlySer (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr Tyr (Y) Trp, Phe, Thr,Ser Phe Val (V) Ile, Leu, Met, Phe, Ala, Leu norleucine

It is to be understood herein, that if a “range” or “group” ofsubstances (e.g. amino acids), substituents” or the like is mentioned orif other types of a particular characteristic (e.g. temperature,pressure, chemical structure, time, etc.) is mentioned, the presentinvention relates to and explicitly incorporates herein each and everyspecific member and combination of sub-ranges or sub-groups thereinwhatsoever. Thus, any specified range or group is to be understood as ashorthand way of referring to each and every member of a range or groupindividually as well as each and every possible sub-ranges or sub-groupsencompassed therein; and similarly with respect to any sub-ranges orsub-groups therein. Thus, for example, with respect to a percentage (%)of identity of from about 80 to 100%, it is to be understood asspecifically incorporating herein each and every individual %, as wellas sub-range, such as for example 80%, 81%, 84.78%, 93%, 99% etc.; andsimilarly with respect to other parameters such as, concentrations,elements, etc. . . .

It is in particular to be understood herein that the methods of thepresent invention each include each and every individual steps describedthereby as well as those defined as positively including particularsteps or excluding particular steps or a combination thereof; forexample an exclusionary definition for a method of the presentinvention, may read as follows: “provided that said polynucleotide doesnot comprise or consist in SEQ ID NO.:57 or the open reading frame ofSEQ ID NO.:57” or “provided that said polypeptide does not comprise orconsist in SEQ ID NO.:100” or “provided that said polynucleotidefragment or said polypeptide fragment is less than X unit (e.g.,nucleotides or amino acids) long or more than X unit (e.g., nucleotidesor amino acids) long”.

Other objects, features, advantages, and aspects of the presentinvention will become apparent to those skilled in the art from thefollowing description. It should be understood, however, that thefollowing description and the specific examples, while indicatingpreferred embodiments of the invention, are given by way of illustrationonly. Various changes and modifications within the spirit and scope ofthe disclosed invention will become readily apparent to those skilled inthe art from reading the following description and from reading theother parts of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

In the appended drawings:

FIG. 1 is a picture of a Southern blot hybridization analysis of the“STAR” subtracted libraries with probes specific for MMP-9, TRAP andGAPDH demonstrating RNA subtraction using STAR;

FIG. 2 shows a pie chart annotation of the clones isolated and sequencedfrom the SL22 subtracted library;

FIG. 3 is a picture illustrating the representative macroarray resultsof osteoclast specificity of the differentially expressed sequencesselected for biological validation;

FIG. 4A is a picture showing the phenotypic effect on osteoclastdifferentiation in the presence of siRNAs specific for SEQ. ID. NO:1;

FIG. 4B is a picture showing the phenotypic effect on osteoclastdifferentiation in the presence of a mixture of siRNAs specific for SEQ.ID. NO:1;

FIG. 5 is a picture of a Northern blot showing the attenuation of SEQ.ID. NO:1 gene expression in RAW cells in the presence of specific siRNAscompared to osteoclast-specific marker genes, TRAP and CTSK;

FIG. 6 are pictures illustrating the phenotypic effect on osteoclastdifferentiation in the presence of siRNAs specific for SEQ. ID. NO:2,panel a; control RAW-hU6 cells, panel b; RAW-hU6 treated with RANKligand, panel c; RAW-hU6 treated with RANK ligand and siRNA specific forSEQ ID NO.:2 (RAW-0179.1), panel d; RAW-hU6 treated with RANK ligand andsiRNA specific for SEQ ID NO.:2 (RAW-0179.2) and panel e; RAW-hU6treated with RANK ligand and siRNA specific for SEQ ID NO.:2(RAW-0179.3);

FIG. 7 is a picture of a Northern blot showing the attenuation of SEQ.ID. NO:2 gene expression in RAW cells in the presence of specificsiRNAs. Also shown is the effect on the osteoclast-specific markergenes, TRAP and Cathepsin K;

FIG. 8 are pictures illustrating the phenotypic effect on osteoclastdifferentiation in the presence of siRNAs specific for SEQ. ID. NO:3panel a; control RAW-hU6 cells, panel b; RAW-hU6 treated with RANKligand, panel c; RAW-hU6 treated with RANK ligand and siRNA specific forSEQ ID NO.:3 (RAW-0799.1), panel d; RAW-hU6 treated with RANK ligand andsiRNA specific for SEQ ID NO.:3 (RAW-0799.2) and panel e; RAW-hU6treated with RANK ligand and siRNA specific for SEQ IDNO.:3(RAW-0799.3);

FIG. 9 are pictures illustrating the phenotypic effect on osteoclastdifferentiation in the presence of siRNAs specific for SEQ. ID. NO:4panel a; control RAW-hU6 cells, panel b; RAW-hU6 treated with RANKligand, and panel c; RAW-hU6 treated with RANK ligand and siRNA specificfor SEQ ID NO.:4 (RAW-0351.1);

FIG. 10 are pictures illustrating the phenotypic effect on osteoclastdifferentiation in the presence of siRNAs specific for SEQ. ID. NO:5panel a; control RAW-hU6 cells, panel b; RAW-hU6 treated with RANKligand, and panel c; RAW-hU6 treated with RANK ligand and siRNA specificfor SEQ ID NO.:5 (RAW-01035.1);

FIG. 11A are pictures illustrating the phenotypic effect on osteoclastdifferentiation in the presence of siRNAs specific for SEQ. ID. NO:6panel a; control RAW-hU6 cells, panel b; RAW-hU6 treated with RANKligand, panel c; RAW-hU6 treated with siRNA specific for SEQ ID NO.:6(RAW-1200.mix) and panel d; RAW-hU6 treated with RANK ligand and siRNAspecific for SEQ ID NO.:6 (RAW-1200.mix);

FIG. 11B are pictures illustrating a time-course of the phenotypiceffect on osteoclast differentiation of RAW-hU6 observed in the presenceor absence of RANK ligand and in the presence or absence siRNAs specificfor SEQ. ID. NO:6 (RAW-1200.mix);

FIG. 12 are pictures illustrating the stimulation of theosteoclast-specific marker genes, TRAP and Cathepsin K, in RAW cellsexpressing specific siRNAs for SEQ. ID. NO:6 in the presence or absenceof RANK ligand.

FIG. 13 are pictures illustrating the phenotypic effect on osteoclastdifferentiation in the presence of siRNAs specific for SEQ. ID. NO:8,panel a; control RAW-hU6 cells, panel b; RAW-hU6 treated with RANKligand, and panel c; RAW-hU6 treated with RANK ligand and siRNA specificfor SEQ ID NO.:8 (RAW-0682.1);

FIG. 14A are pictures illustrating the reduced resorptive activity ofosteoclasts expressing specific siRNAs for SEQ. ID. NO:1 panel a;control RAW-hU6 cells, panel b; RAW-hU6 treated with RANK ligand, panelc; RAW-hU6 treated with RANK ligand and siRNA specific for SEQ ID NO.:1(RAW-0440.1), panel d; RAW-hU6 treated with RANK ligand and siRNAspecific for SEQ ID NO.:1 (RAW-0440.2) and panel e; RAW-hU6 treated withRANK ligand and siRNA specific for SEQ ID NO.:1 (RAW-0440.3);

FIG. 14B is an histogram illustrating the results of FIG. 14A in aquantitative manner;

FIG. 15A shows the reduced resorptive activity of osteoclasts expressingspecific siRNAs for SEQ. ID. NO:2 panel a; control RAW-hU6 cells, panelb; RAW-hU6 treated with RANK ligand, panel c; RAW-hU6 treated with RANKligand and siRNA specific for SEQ ID NO.:2 (RAW-0179.1), panel d;RAW-hU6 treated with RANK ligand and siRNA specific for SEQ ID NO.:2(RAW-0179.2) and panel e; RAW-hU6 treated with RANK ligand and siRNAspecific for SEQ ID NO.:2 (RAW-0179.3);

FIG. 15B is an histogram illustrating the results of FIG. 15A in aquantitative manner;

FIG. 16A is a picture representing an examplary embodiment of amacroarray hybridization results of differential expression of somehuman orthologues in the different human tissues and human osteoclastssamples;

FIG. 16B is a picture of an agarose gel of RT-PCR-amplified SEQ ID NO.:1in human precursor and osteoclast samples;

FIG. 17 is a picture illustrating the phenotypic effect on osteoclastdifferentiation in the presence of siRNAs specific for the humanorthologue for SEQ ID NO.:1 (SEQ. ID. NO:88) right panel; control siRNA,left panel AB0440 siRNA;

FIG. 18 are pictures illustrating the efficiency of the functionalcomplementation assay for SEQ. ID. NO. 88 to screen for inhibitors ofosteoclastogenesis;

FIG. 19 is a picture of a Western blot from cell lysate obtained fromcells expressing a SEQ ID NO.:88 fusion protein and treated or not withtunicamycin or phosphoinositol phospholipase C;

FIG. 20A is an histogram quantifying the inhibition of RAW264.7differentiation into osteoclast using a monoclonal anti-Tsp50 (SEQ. ID.NO. 1) antibody;

FIG. 20B are pictures representing the phenotypic inhibition of RAW264.7differentiation into osteoclast using a monoclonal anti-Tsp50 (SEQ. ID.NO. 1) antibody

FIG. 21A is a picture of a Northern blot illustrating that SEQ. ID. NO.89 (d2) expression is upregulated in osteoclasts compared to the d1isoform,

FIG. 21B are pictures of Western blots of a pull-down assay illustratinginteraction of d2 with the v-ATPase a3 subunit but not the a4 subunitand;

FIGS. 22 to 87 represents some polynucleotides and polypeptidesidentified using an examplary method of the present invention.

SEQ ID NOs: 1-7, and 57 show differentially expressed sequences found inosteoclasts and demonstrated to have an effect on osteoclastogenesisfollowing inhibition with specific siRNAs. SEQ ID NOs: 8-56 showdifferentially expressed sequences found in osteoclasts with putativeroles in bone remodelling. SEQ ID NOs: 58-82 show the nucleotidesequences of plasmids, oligonucleotide primers and siRNAs used forexperiments performed herein.

SEQ ID NOs: 83-87 show the mRNA sequence of spliced variants isolatedfrom RNA prepared from osteoclasts for some of the osteoclast-specificsequences identified. SEQ ID NOs 83-87 thus show by way of examples thatunique spliced variants exist and strongly suggest that others alsoexist for the model system under study and others. More specifically,SEQ ID NO: 83 is a variant of SEQ ID NO: 1; SEQ ID NO: 84 is a humansequence of the corresponding mouse variant #1 for SEQ ID NO: 2; SEQ IDNO: 85 is a human sequence of the corresponding mouse variant #2 for SEQID NO: 2; SEQ ID NO: 86 is a variant of SEQ ID NO: 3; and SEQ ID NO: 87is a variant of SEQ ID NO: 3.

SEQ ID NO.: 88 and 89 shows the mRNA sequence of human orthologs of SEQID NO.:1 and 2 respectively.

SEQ ID NO.: 64 to 82 and 90 shows fragments which are complementary to aportion of a sequence of selected polynucleotides described herein.

SEQ ID NO.: 93 to 155 shows polypeptides encoded by the polynucleotidesof the present invention.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The applicant employed a carefully planned strategy to identify andisolate genetic sequences involved in osteoclastogenesis and boneremodeling. The process involved the following steps: 1) preparation ofhighly representative cDNA libraries using mRNA isolated from precursorsand osteoclasts; 2) isolation of sequences upregulated duringosteoclastogenesis; 3) identification and characterization ofupregulated sequences; 4) selection of upregulated sequences for tissuespecificity; 5) determination of knock-down effects onosteoclastogenesis and 6) determination of knock-down effects on boneresorption.

The results discussed in this disclosure demonstrate the advantage oftargeting osteoclast genes that are specific to this differentiated celltype and provide a more efficient screening method when studying thegenetic basis of diseases and disorders. Genes that are known to have arole in other areas of biology have been shown to play a critical rolein osteoclastogenesis and osteoclast function. Genes that are known buthave not had a role assigned to them until the present disclosure havealso been isolated and shown to have a critical role inosteoclastogenesis and osteoclast function. Finally, novel genes havebeen identified and play a role, however, applicant reserves theirdisclosure until further study has been completed.

The present invention is illustrated in further details below in anon-limiting fashion.

A—Material and Methods

Commercially available reagents referred to in the present disclosurewere used according to supplier's instructions unless otherwiseindicated. Throughout the present disclosure certain starting materialswere prepared as follows:

B—Preparation of Osteoclast Differentiated Cells

The RAW 264.7 (RAW) osteoclast precursor cell line and human CD34+progenitors are well known in the art as murine and human models ofosteoclastogenesis. These murine and human osteoclasts are thereforeexcellent sources of materials for isolating and characterizing genesspecialized for osteoclast function.

RAW cells were purchased from American Type Culture Collection andmaintained in high glucose DMEM containing 10% fetal bovine serum andantibiotics. The cells were sub-cultured bi-weekly to a maximum of 10-12passages. For osteoclast differentiation experiments, RAW cells wereseeded in 96-well plates at a density of 4×103 cells/well and allowed toplate for 24 h. Differentiation was induced in high glucose DMEM, 10%charcoal-treated foetal bovine serum (Hyclone, Logan, Utah), 0.05% BSA,antibiotics, 10 ng/ml macrophage colony stimulating factor (M-CSF), and100 ng/ml receptor activator of NF-kB (RANK) ligand. The plates werere-fed on day 3 and osteoclasts were clearly visible by day 4.Typically, the cells were stained for tartrate-resistant acidphosphatase (TRAP) on day 4 or 5 unless otherwise indicated. For TRAPstaining, the cells were washed with PBS and fixed in 10% formaldehydefor 1 h. After two PBS washes, the cells were rendered lightly permeablein 0.2% Triton X-100 in PBS for 5 min before washing in PBS. Stainingwas conducted at 37° C. for 20-25 min in 0.01% Naphtol AS-MX phosphate,0.06% Fast Red Violet, 50 mM sodium tartrate, 100 mM sodium acetate, pH5.2. Cells were visualized microscopically.

Human osteoclasts were differentiated from G-CSF-mobilized peripheralblood mononuclear cells (Cambrex, East Rutherford, N.J.) as described bythe supplier in the presence of 35 ng/ml M-CSF and 100 ng/ml RANKligand. Multinucleated TRAP-staining osteoclasts were visible by 11-14days. Osteoclasts from human cells were also derived from humanosteoclasts precursor cells (Cambrex, East Rutherford, N.J.) andcultured as described by the supplier. In the latter case, osteoclastsare obtained after 7 days.

C—Method of Isolating Differentially Expressed mRNA

Key to the discovery of differentially expressed sequences unique toosteoclasts is the use of the applicant's patented STAR technology(Subtractive Transcription-based Amplification of mRNA; U.S. Pat. No.5,712,127 Malek et al., Jan. 27, 1998). In this procedure, mRNA isolatedfrom fully differentiated osteoclasts is used to prepare “tester RNA”,which is hybridized to complementary single-stranded “driver DNA”prepared from osteoclast precursor mRNA and only the un-hybridized“tester RNA” is recovered, and used to create cloned cDNA libraries,termed “subtracted libraries”. Thus, the “subtracted libraries” areenriched for differentially expressed sequences inclusive of rare andnovel mRNAs often missed by micro array hybridization analysis, whichare anticipated to be among the important gene targets for thedevelopment of better diagnostic and therapeutic strategies.

The clones contained in the enriched “subtracted libraries” areidentified by DNA sequence analysis and their potential functionassessed by database analysis. The non-redundant clones are then used toprepare DNA micro-arrays, which are used to quantify their relativedifferential expression patterns by hybridization to fluorescent cDNAprobes. Two classes of cDNA probes are used, which are generated fromeither RNA transcripts prepared from the same subtracted libraries(subtracted probes) or mRNA isolated from different osteoclast samples(standard probes). The use of subtracted probes provides increasedsensitivity for detecting the low abundance mRNA sequences that arepreserved and enriched by STAR. Furthermore, the specificity of thedifferentially expressed sequences to osteoclast is measured byhybridizing radio-labeled probes prepared from each selected sequence tomacroarrays containing RNA from different osteoclast samples anddifferent murine and/or human tissues. Additionally, Northern blotanalysis is performed so as to confirm the presence of one or morespecific mRNA species in the osteoclast samples. Following this, thefull-length cDNAs representative of the mRNA species and/or splicedvariants are cloned in E. coli DH10B.

A major challenge in gene expression profiling is the limited quantitiesof RNA available for molecular analysis. The amount of RNA isolated frommany osteoclast samples or human specimens (needle aspiration, lasercapture micro-dissection (LGM) samples and transfected cultured cells)is often insufficient for preparing: 1) conventional tester and drivermaterials for STAR; 2) standard cDNA probes for DNA micro-arrayanalysis; 3) RNA macroarrays for testing the specificity of expression;4) Northern blots and; 5) full-length cDNA clones for further biologicalvalidation and characterization. Thus, the applicant has developed aproprietary technology called RAMP (RNA Amplification Procedure) (U.S.patent application Ser. No. 11/000,958 published under No. US2005/0153333A1 on Jul. 14, 2005 and entitled “Selective Terminal Taggingof Nucleic Acids”), which linearly amplifies the mRNA contained in totalRNA samples yielding microgram quantities of amplified RNA sufficientfor the various analytical applications. The RAMP RNA produced islargely full-length mRNA-like sequences as a result of the proprietarymethod for adding a terminal sequence tag to the 3′-ends ofsingle-stranded cDNA molecules, for use in linear transcriptionamplification. Greater than 99.5% of the sequences amplified in RAMPreactions show <2-fold variability and thus, RAMP provides unbiased RNAsamples in quantities sufficient to enable the discovery of the uniquemRNA sequences involved in osteoclastogenesis.

D—Preparation of Murine Osteoclasts Subtracted Library

RAW precursor cells and the corresponding fully differentiated (day 5)osteoclasts were prepared as described above. Isolation of cellular RNAfollowed by mRNA purification from each was performed using standardmethods (Qiagen, Mississauga, ON). Following the teachings of Malek etal. (U.S. Pat. No. 5,712,127), 2 μg of poly A+ mRNA from each samplewere used to prepare highly representative (>2×10⁶ CFU) cDNA librariesin specialized plasmid vectors necessary for preparing tester and drivermaterials. In each case, first-strand cDNA was synthesized using anoligo dT₁₁ primer with 3′ locking nucleotides (e.g., A, G or C) andcontaining a Not I recognition site. Next, second-strand cDNA synthesiswas performed according to the manufacturer's procedure fordouble-stranded cDNA synthesis (Invitrogen, Burlington, ON) and theresulting double-stranded cDNA ligated to linkers containing an Asc Irecognition site (New England Biolabs, Pickering, ON). Thedouble-stranded cDNAs were then digested with Asc I and Not Irestriction enzymes (New England Biolabs, Pickering, ON), purified fromthe excess linkers using the cDNA fractionation column from Invitrogen(Burlington, ON) as specified by the manufacturer and each ligated intospecialized plasmid vectors—p14 (SEQ. ID. NO:58) and p17+ (SEQ. ID.NO:59) used for preparing tester and driver materials respectively.Thereafter, the ligated cDNAs were transformed into E. coli DH10Bresulting in the desired cDNA libraries (RAW 264.7-precursor-p14, RAW264.7-precursor-p17+, RAW 264.7-osteoclasts-p14 and RAW264.7-osteoclasts-p17+). The plasmid DNA pool for each cDNA library waspurified and a 2-μg aliquot of each linearized with Not I restrictionenzyme. In vitro transcription of the Not I digested p14 and p17+plasmid libraries was then performed with T7 RNA polymerase and sp6 RNApolymerase respectively (Ambion, Austin, Tex.).

Next, in order to prepare 3′-represented tester and driver libraries, a10-μg aliquot of each of the in vitro synthesized RNA was converted todouble-stranded cDNA by performing first-strand cDNA synthesis asdescribed above followed by primer-directed (primer OGS 77 for p14 (SEQ.ID. NO:62) and primer OGS 302 for p17+ (SEQ. ID. NO:63)) second-strandDNA synthesis using Advantage-2 Taq polymerase (BD Biosciences Clontech,Mississauga, ON). The sequences corresponding to OGS 77 and OGS 302 wereintroduced into the in vitro synthesized RNA by way of the specializedvectors used for preparing the cDNA libraries. Thereafter, 6× 1-μgaliquots of each double-stranded cDNA was digested individually with oneof the following 4-base recognition restriction enzymes Rsa I, Sau3A1,Mse I, Msp I, MinPl I and Bsh 1236I (MBI Fermentas, Burlington, ON),yielding up to six possible 3′-fragments for each RNA species containedin the cDNA library. Following digestion, the restriction enzymes wereinactivated with phenol and the set of six reactions pooled. Therestriction enzymes sites were then blunted with T4 DNA polymerase andligated to linkers containing an Asc I recognition site. Eachlinker-adapted pooled DNA sample was digested with Asc I and Not Irestriction enzymes, desalted and ligated to specialized plasmidvectors, p14 and p17 (p17 plasmid vector is similar to the p17+ plasmidvector except for the sequence corresponding to SEQ. ID. NO:63), andtransformed into E. coli DH10B. The plasmid DNA pool for each p14 andp17 3′-represented library was purified (Qiagen, Mississauga, ON) and a2-mg aliquot of each digested with Not I restriction enzyme, andtranscribed in vitro with either T7 RNA polymerase or sp6 RNA polymerase(Ambion, Austin, Tex.). The resulting p14 3′-represented RNA was useddirectly as “tester RNA” whereas, the p17 3′-represented RNA was used tosynthesize first-strand cDNA as described above, which then served as“driver DNA”. Each “driver DNA” reaction was treated with RNase A andRNase H to remove the RNA, phenol extracted and desalted before use.

The following 3′-represented libraries were prepared:

-   -   Tester 1—RAW 264.7-osteoclast-3′ in p14    -   Tester 2—RAW 264.7-precursor-3′ in p14    -   Driver 1—RAW 264.7-precursor-3′ in p17    -   Driver 2—RAW 264.7-osteoclast-3′ in p17

The tester RNA samples were subtracted following the teachings of U.S.Pat. No. 5,712,127 with the corresponding driver DNA in a ratio of 1:100for either 1- or 2-rounds following the teachings of Malek et al. (U.S.Pat. No. 5,712,127). Additionally, control reactions containing testerRNA and no driver DNA, and tester RNA plus driver DNA but no RNase Hwere prepared. The tester RNA remaining in each reaction aftersubtraction was converted to double-stranded DNA, and 5% removed andamplified in a standard PCR reaction for 30-cycles for analyticalpurposes. The remaining 95% of only the driver plus RNase H subtractedsamples were amplified for 4-cycles in PCR, digested with Asc I and NotI restriction enzymes, and one half ligated into the pCATRMAN (SEQ. ID.NO:60) plasmid vector and the other half, into the p20 (SEQ. ID. NO:61)plasmid vector. The ligated materials were transformed into E. coliDH10B and individual clones contained in the pCATRMAN libraries werepicked for further analysis (DNA sequencing and hybridization) whereas,clones contained in each p20 library were pooled for use as subtractedprobes. Each 4-cycles amplified cloned subtracted library containedbetween 25,000 and 40,000 colonies.

The following cloned subtracted libraries were prepared:

-   -   T04-22—tester 1 (osteoclast) minus driver 1 (precursor)        (1-round) in pCATRMAN    -   SL22—tester 1 (osteoclast) minus driver 1 (precursor) (2-rounds)        in pCATRMAN    -   SL22—tester 1 (osteoclast) minus driver 1 (precursor) (2-rounds)        in p20    -   SL27—tester 2 (precursor) minus driver 2 (osteoclast) (2-rounds)        in pCATRMAN    -   SL27—tester 2 (precursor) minus driver 2 (osteoclast) (2-rounds)        in p20

A 5-μL aliquot of the 30-cycles PCR amplified subtracted materialsdescribed above were visualized on a 1.5% agarose gel containingethidium bromide and then transferred to Hybond N+ (AmershamBiosciences, Piscataway, N.J.) nylon membrane for Southern blotanalysis. Three identical Southern transfers were prepared and werehybridized separately to radiolabeled probes specific to the MMP-9(matrix metalloproteinase 9; NM_(—)013599.2) and TRAP (tartrateresistant acid phosphatase; NM_(—)007388.1) genes, which are known to beupregulated in osteoclasts, and GAPDH (glyceraldehyde-3-phosphatedehydrogenase; M32599.1), which is a non-differentially expressedhouse-keeping gene. The results of the hybridization analysis are shownin FIG. 1 where the following lanes contain the following materials:

-   -   AO 1—tester 1 RNA plus driver 1 DNA plus RNase H (1-round)    -   AO 2—tester 1 RNA plus driver 1 DNA plus RNase H (2-rounds)    -   AP 1—tester 2 RNA plus driver 2 DNA plus RNase H (1-round)    -   AP 2—tester 2 RNA plus driver 2 DNA plus RNase H (2-rounds)    -   BO 1—tester 1 RNA plus driver 1 DNA minus RNase H (1-round)    -   BO 2—tester 1 RNA plus driver 1 DNA minus RNase H (2-rounds)    -   BP 1—tester 2 RNA plus driver 2 DNA minus RNase H (1-round)    -   BP 2—tester 2 RNA plus driver 2 DNA minus RNase H (2-rounds)    -   CO 1—tester 1 RNA minus driver 1 DNA plus RNase H (1-round)    -   CO 2—tester 1 RNA minus driver 1 DNA plus RNase H (2-rounds)    -   CP 1—tester 2 RNA minus driver 2 DNA plus RNase H (1-round)    -   CP 2—tester 2 RNA minus driver 2 DNA plus RNase H (2-rounds)    -   DP—tester 2 RNA    -   DO—tester 1 RNA

These results clearly show reduction of the GAPDH mRNA levels,representative of a non-differentially expressed gene, when both driverDNA and RNase H were present in the reactions (complete) (GAPDH panel:Lanes AO 1, AO 2, AP 1 and AP 2) in comparison to the incompletereactions (GAPDH panel: BO 1, BO 2, BP 1, BP 2, CO 1, CO 2, CP 1 and CP2). Additionally, there was better subtraction of GAPDH after 2-rounds(GAPDH panel: AO 2 and AP 2) compared to 1-round (GAPDH panel: AO 1 andAP 1). On the other hand, the differentially expressed upregulated genes(MMP-9 and TRAP) were enriched in the complete reactions (MMP-9 and TRAPpanels: Lanes AO 1 and AO 2) in comparison to the incomplete reactions(MMP-9 and TRAP panels: BO 1, BO 2, CO 1 and CO 2), which showed amountssimilar to the intact tester RNA (MMP-9 and TRAP panels: Lane DO).

Based on these results, it was anticipated that the subtracted librarieswould be enriched for differentially expressed sequences. Thus, forT04-22 and SL22 libraries, genes up regulated in osteoclasts would berepresented whereas, for SL27, the down-regulated genes would berepresented.

E—Sequence Identification and Annotation of Clones Contained in theT04-22 and SL22 Subtracted Libraries:

Since GAPDH (see above) was most efficiently subtracted after 2-rounds(SL-22), it was anticipated that this library would be most enriched fordifferentially expressed osteoclast-related sequences. Thus, moreexhaustive DNA sequence analysis was performed on clones contained inSL22 (1536 clones) compared to T04-22 (576 clones).

The individual colonies contained in the T04-22- and SL22-pCATRMANlibraries prepared as described previously were randomly picked using aQbot (Genetix Inc., Boston, Mass.) into 60 μL of autoclaved water. Then,42 μL of each was used in a 100-μL standard PCR reaction containingoligonucleotide primers, OGS 1 and OGS 142 and amplified for 40-cycles(94° C. for 10 minutes, 40× (94° C. for 40 seconds, 55° C. for 30seconds and 72° C. for 2 minutes) followed by 72° C. for 7 minutes) in96-wells microtitre plates using HotStart™ Taq polymerase (Qiagen,Mississauga, ON). The completed PCR reactions were desalted using the96-well filter plates (Corning) and the amplicons recovered in 100 μL 10mM Tris (pH 8.0). A 5-μL aliquot of each PCR reaction was visualized ona 1.5% agarose gel containing ethidium bromide and only those reactionscontaining a single amplified product were selected for DNA sequenceanalysis using standard DNA sequencing performed on an ABI 3100instrument (Applied Biosystems, Foster City, Calif.). Each DNA sequenceobtained was given a Sequence Identification Number and entered into adatabase for subsequent tracking and annotation.

For the purpose of illustrating the ensuing strategies foridentification of the clones, only the DNA sequences obtained for clonescontained in SL22 will be discussed further. Of those sequences, 1408were selected for BLAST analysis of public databases (e.g. NCBI), whichyielded 744 unique sequences representing a redundancy of approximately53% and thus, a sufficiently representative sampling of the subtractedlibrary. Absent from these sequences were the standard housekeepinggenes (GAPDH, actin, most ribosomal proteins etc.), which was a goodindication that the subtracted library was depleted of at least therelatively abundant non-differentially expressed sequences. A limitedsurvey of 96 clones from a corresponding un-subtracted library resultedin largely known and abundant housekeeping sequences such as GAPDH andbeta-actin (data not shown). The 744 unique sequences were broadlyclassified into three categories shown in FIG. 2: 522 genes with Unigeneclusters (70.2%), 84 genes with no Unigene cluster (11.3%) and 138 novelsequences (18.5%). Of the Unigene-clustered genes, only 114 wereassociated with GO (Gene Ontology) functional categories. Thus, it wasevident from these results that the subtracted library (SL22) wasenriched for known and novel sequences.

Once sequencing and annotation of the selected clones were completed,the next step involved identifying those sequences that were actuallyupregulated in osteoclasts compared to precursors.

F—Hybridization Analysis for Identifying Upregulated Sequences

The PCR amplicons representing the annotated sequences from the T04-22and SL22 libraries were used to prepare DNA microarrays. The purifiedPCR amplicons from contained in 70 μL prepared in the previous sectionwas lyophilized and each reconstituted in 20 μL of spotting solutioncomprising 3×SSC and 0.1% sarkosyl. DNA micro-arrays of each amplicon intriplicate were then prepared using CMT-GAP2 slides (Corning, Corning,N.Y.) and the GMS 417 spotter (Affymetrix, Santa Clara, Calif.).

The DNA micro-arrays were then hybridized with either standard orsubtracted cy3 and cy5 labelled cDNA probes as recommended by thesupplier (Amersham Biosciences, Piscataway, N.J.). The standard cDNAprobes were synthesized using RAMP amplified

RNA prepared from five different murine osteoclast samples and thecorresponding precursors. It is well known to the skilled artisan thatstandard cDNA probes only provide limited sensitivity of detection andconsequently, low abundance sequences contained in the cDNA probes areusually missed. Thus, the hybridization analysis was also performedusing subtracted cDNA probes. These subtracted cDNA probes weresynthesized from in vitro transcribed RNA prepared from the SL22-p20 andSL25-p20 subtracted libraries described above in D. These subtractedlibraries may be enriched for low abundance sequences as a result offollowing the teachings of Malek et al., and therefore, may provideincreased detection sensitivity.

All hybridization reactions were performed using the dye-swap procedureas recommended by the supplier (Amersham Biosciences, Piscataway, N.J.).Following analysis of the hybridization results obtained using thestandard cDNA probes, 161 of the 744 unique sequences contained in SL22appeared to be upregulated in the osteoclasts, showing >2-folddifference compared to the precursors. On the other hand, when thesubtracted cDNA probes were used, 289 additional SL22-sequences appearedto be upregulated in the osteoclast samples as well.

Thus, it was evident from these results that the SL22 subtracted librarywas highly enriched for upregulated sequences (>60%), which wereprobably involved in osteoclastogenesis. A similar analysis wasperformed for the T04-22 clones, which showed a lower percentage ofdifferentially expressed sequences likely due to insufficientsubtraction after only 1-round of the STAR procedure.

G—Determining Osteoclast Specificity of the Differentially ExpressedSequences Identified:

The differentially expressed sequences identified in Section F for bothSL22 and T04-22 libraries were tested for osteoclast specificity byhybridization to nylon membrane-based macroarrays. The macroarrays wereprepared using RAMP amplified RNA from murine precursors and osteoclastsof five independent experiments, and various normal murine tissues(liver, brain, thymus, heart, lung, testicule, ovary, kidney and embryo)purchased commercially (Ambion, Austin, Tex.). Because of the limitedquantities of mRNA available for many of these samples, it was necessaryto first amplify the mRNA using the RAMP methodology. Each amplified RNAsample was reconstituted to a final concentration of 250 ng/μL in 3×SSCand 0.1% sarkosyl in a 96-well microtitre plate and 1 μL spotted ontoHybond N+ nylon membranes using the specialized MULTI-PRINT™ apparatus(VP Scientific, San Diego, Calif.), air dried and UV-cross linked. Atotal of 556 different sequences selected from SL22 and T04-22 wereindividually radiolabeled with α-³²P-dCTP using the random primingprocedure recommended by the supplier (Amersham, Piscataway, N.J.) andused as probes on the macroarrays. Hybridization and washing steps wereperformed following standard procedures well known to those skilled inthe art.

Of the 556 sequences tested, approximately 80% were found to beupregulated in at least the primary osteoclast RNA sample that was usedto prepare the subtracted libraries. However, many of these sequenceswere also readily detected in the different murine tissues. Based onthese results, those sequences that appeared to be associated withexperimental variability and those that were detected in many of theother murine tissues were eliminated. Consequently, only 73 sequences,which appeared to be highly osteoclast-specific, were selected forbiological validation studies. This subset of 73 sequences includedsequences present in two or less murine tissues relative to theprecursor levels since it is entirely possible that the hybridizationsignals obtained for these tissues maybe due to family members orspliced variants.

FIG. 3 shows examples of the macroarray patterns representative of thesequences selected for validation. Subsequently, RNA from 8 additionalnormal murine tissues (lymph node, eye, prostate, smooth muscle, spinalcord, stomach, uterus and bone marrow) were incorporated into secondarymacroarrays and used to further test the specificity of many of the 73selected sequences (data not shown). Amongst the 73 selected sequenceswere 41 genes with functional annotation of which, only two werepreviously linked to osteoclastogenesis (Unigene Clusters Mm.103560 andMm.271689), 20 genes with no functional annotation and 12 novelsequences (data not disclosed). Representative sequences arecharacterized as follows:

SEQ. ID. NO:1:

The candidate protein encoded by SEQ. ID. NO:1 is a previouslyidentified gene with the designation, testis-specific protease or Tsp50.The mouse polynucleotide contains an open reading frame of 1317 bp andencodes a polypeptide of 439 amino acids. The human polynucleotidecontains an open reading frame of 1155 bp and encodes a polypeptide of385 amino acids. It was originally described and cloned because of itsexpression in a hypomethylated region of genomic DNA in human breastcancer cells (Yuan et al., 1999). Analysis of the primary amino acidsequence suggests the presence of an amino-terminal signal peptide thatwill presumably target the protein to the plasma membrane, acarboxy-terminal transmembrane domain to anchor the protein in theplasma membrane, and a predicted catalytic domain homologous to serineproteases (Shan et al., 2002; Netzel-Arnett et al., 2003). The nature ofthe predicted catalytic activity and the exact cellular localization ofTsp50 have yet to be conclusively established. Applicant directs thereader's attention to U.S. Pat. No. 6,617,434 (Duffy, Sep. 9, 2003) andU.S. Pat. No. 6,451,555 (Duffy, Sep. 17, 2002) where Tsp50 is thesubject matter. Despite all of the above information, no functionalassociation with osteoclasts or bone remodeling disorders has beendescribed prior to the present disclosure.

SEQ. ID. NO:2:

The candidate protein encoded by SEQ. ID. NO:2 is a previouslyidentified gene that encodes the d2 subunit of the vacuolar (V-) ATPasemulti-subunit complex. Although the d2 subunit does not span themembrane, it is part of the membrane-spanning complex and interactsdirectly with the larger a subunit that contains the transmembraneproperties (Nishi and Forgac, 2002). The cDNA encoding the mouseV-ATPase d2 protein has recently been described but its function in bonephysiology has yet to be established (Nishi et al., 2003). No functionalassociation with osteoclasts or bone remodeling disorders has beendescribed prior to the present disclosure.

SEQ. ID. NO:3:

The candidate protein encoded by SEQ. ID. NO:3 is a previouslyidentified gene that encodes the cartilage-associated protein, Crtap(Morello et al., 1999; Tonachini et al., 1999). The gene was originallycloned from chick embryo and localized to cartilaginous tissues (Morelloet al., 1999). No functional association with osteoclasts or boneremodeling disorders has been described prior to the present disclosure.

SEQ. ID. NO:4:

The candidate protein encoded by SEQ. ID. NO:4 is found in currentdatabases and was cloned as part of the RIKEN Genome ExplorationResearch Group (Kawai et al., 2001). Although the mRNA contains apredicted open reading frame, no function has been assigned to thissequence prior to the present disclosure.

SEQ. ID. NO:5:

The candidate protein encoded by SEQ. ID. NO:5 is found in currentdatabases and was cloned as part of the RIKEN Genome ExplorationResearch Group (Strausberg et al., 2002). Although the mRNA contains apredicted open reading frame, no function has been assigned to thissequence prior to the present disclosure.

SEQ. ID. NO:6:

The candidate protein encoded by SEQ. ID. NO:6 is found in currentdatabases and was cloned as part of the RIKEN Genome ExplorationResearch Group (Strausberg et al., 2002). Although the mRNA contains apredicted open reading frame, no function has been assigned to thissequence prior to the present disclosure.

SEQ. ID. NO:7:

The candidate protein encoded by SEQ. ID. NO:7 is a previouslyidentified gene that encodes the linker for activation of B cells. Thegene has been reported as playing a role in thymocytes (Janssen et al.,2003). No functional association with osteoclasts or bone resorptiondisorders has been described prior to the present disclosure.

SEQ. ID. NO:57

The candidate protein encoded by SEQ. ID. NO:57 is a previouslyidentified gene that encodes the jun dimerization protein 2. This genehas been shown to be involved in osteoclastogenesis using antisensetechnology (Kawaida et al., 2003). This example serves as further proofof concept of applicant's approach in identifying osteoclast-specificgenes.

H—Cloning of Full-Length cDNAs of Selected Sequences from OsteoclastmRNA:

It was necessary to obtain full-length cDNA sequences in order toperform functional studies of the expressed proteins. Spliced variantsare increasingly being implicated in tissue specific functions and assuch, it is critically necessary to work with cDNA clones from thesystem under study. Applicant also recognizes that spliced variants maynot always be involved. Thus, the applicant's approach has been toisolate the relevant full-length cDNA sequences directly fromosteoclasts in order to identify variants and their potential role withrespect to specificity.

Coding cDNA clones were isolated using both a 5′-RACE strategy(Invitrogen, Burlington, ON) and a standard two-primer gene specificapproach in PCR. The 5′-RACE strategy used cDNA prepared fromcap-selected osteoclast RNA and/or RAMP amplified osteoclast RNA. Foramplification using gene specific primers, either cDNA prepared fromRAMP RNA or total RNA was used. All cDNAs were synthesized followingstandard reverse transcription procedures (Invitrogen, Burlington, ON).The cDNA sequences obtained were cloned in E. coli DH10B and thenucleotide sequences for multiple clones determined. Thereafter, thecDNA sequences for each set were aligned and the open reading frame(s)(ORF) identified using standard software (e.g. ORF Finder-NCBI). Table 3shows examples of cDNA clones of spliced variants, which were obtainedfor some of the sequences under investigation.

I—RNA Interference Studies

RNA interference is a recently discovered gene regulation mechanism thatinvolves the sequence-specific decrease in a gene's expression bytargeting the mRNA for degradation and although originally described inplants, it has been discovered across many animal kingdoms fromprotozoans and invertebrates to higher eukaryotes (reviewed in Agrawalet al., 2003). In physiological settings, the mechanism of RNAinterference is triggered by the presence of double-stranded RNAmolecules that are cleaved by an RNAse III-like protein active in cells,called Dicer, which releases the 21-23 bp siRNAs. The siRNA, in ahomology-driven manner, complexes into a RNA-protein amalgamation termedRISC (RNA-induced silencing complex) in the presence of mRNA to causedegradation resulting in attenuation of that mRNA's expression (Agrawalet al., 2003).

Current approaches to studying the function of genes, such as geneknockout mice and dominant negatives, are often inefficient, andgenerally expensive, and time-consuming. RNA interference is proving tobe a method of choice for the analysis of a large number of genes in aquick and relatively inexpensive manner. Although transfection ofsynthetic siRNAs is an efficient method, the effects are often transientat best (Hannon G. J., 2002). Delivery of plasmids expressing shorthairpin RNAs by stable transfection has been successful in allowing forthe analysis of RNA interference in longer-term studies (Brummelkamp etal., 2002; Elbashir et al., 2001). In addition, more recent advanceshave permitted the expression of siRNA molecules, in the form of shorthairpin RNAs, in primary human cells using viral delivery methods suchas lentivirus (Lee et al., 2004; Rubinson et al., 2003).

J—Determination of Knockdown Effects on Osteoclastogenesis

The design and subcloning of individual siRNA expression cassettes andthe procedure utilized for the characterization of each nucleotidesequence is described below. Selection of polynucleotides were chosenbased on their RANK ligand-dependent upregulation in osteoclasts and theselective nature of their expression in osteoclasts compared to othertissues (see sections F and G above). The design of siRNA sequences wasperformed using web-based software that is freely available to thoseskilled in the art (Qiagen for example). These chosen sequences, usually19-mers, were included in two complimentary oligonucleotides that formthe template for the short hairpin RNAs, i.e. the 19-nt sense sequence,a 9-nt linker region (loop), the 19-nt antisense sequence followed by a5-6 poly-T tract for termination of the RNA polymerase III. Appropriaterestriction sites were inserted at the ends of these oligonucleotides tofacilitate proper positioning of the inserts so that the transcriptionalstart point is at a precise location downstream of the hU6 promoter. Foreach sequence selected, at least two different siRNA expression vectorswere constructed to increase the chance of observing RNA interference.

The transfection plasmids expressing the siRNAs under the control of thehuman U6 promoter were constructed as follows. Two primers containing anAseI site (forward) and a KpnI site (reverse) were used to PCR amplify a330-bp fragment containing the human U6 promoter from 5 ng of humangenomic DNA. This fragment was ligated in similarly digested pd2EGFP-N1(BD Biosciences Clontech, Mississauga, ON) resulting in the replacementof the CMV E1 promoter of pd2EGFP-N1 by the human (h)U6 promotersequence. Digesting with AgeI and NotI and religating the blunted endsto generate pd2-hU6 accomplished removal of the d2EGFP fragment. Thetemplate for the siRNA hairpin was designed by annealing twooligonucleotides yielding a 57-bp fragment blunt at the 5′-end andhaving a BamHI overhang at the 3′-end. The annealed oligonucleotideswere ligated into pd2-hU6 that had been previously digested with KpnI(blunted) and BamHI resulting in pd2-hU6/siRNA. All plasmids wereverified by sequencing to confirm presence of the siRNA hairpin sequenceand proper positioning of the transcriptional start site following thehU6 promoter.

RAW cells were seeded in 6-well plates in high glucose DMEM containing10% fetal bovine serum at a density of 6×10⁵ cells/well, allowed toplate overnight and transfected with 1 μg of pd2-hU6/siRNA plasmid usingthe Fugene 6 reagent (Roche, Laval, QC). After 16 h of incubation, freshmedium was added containing 400 μg/ml G418 to select for stabletransfectants. Control cells were transfected with pd2-hU6. Afterapproximately 10 days, pools and/or individual clones of cells wereisolated and analyzed for their ability to form osteoclasts in 96-wellplates. The resulting phenotypes were observed microscopically byviewing cells assayed for TRAP staining. The efficacy of RNAinterference was also assessed by conducting Northern blots on total RNAisolated from cells subjected to in vitro osteoclastogenesis.

K—Results of RNA Interference Studies

SEQ. ID. NO:1:

The sequences used for RNA interference were derived from thepolynucleotide SEQ. ID. NO:1 and have the SEQ. ID. NOs: 64, 65 and 66(of course other sequences may be used). The cell lines derived from RAWcells transfected with plasmids encoding the three siRNAspd2-hU6/0440.1, pd2-hU6/0440.2 and, pd2-hU6/0440.3 are designatedhereafter as RAW-0440.1 (SEQ. ID. NO.:64), RAW-0440.2 (SEQ. ID. NO.:65), and RAW-0440.3 (SEQ. ID. NO.:66), respectively and collectively asRAW-0440. In addition, as a positive control for normalosteoclastogenesis, RAW cells were transfected with the empty vector(pd2-hU6) that does not contain a siRNA. Phenotypic analysis of all celllines is shown in FIG. 4A. Panel a of FIG. 4A shows the control cellline, RAW-hU6, in the absence of RANK ligand where the presence ofmultinucleated osteoclasts is not observed and the undifferentiated RAWcells are completely devoid of TRAP staining. Upon treatment with RANKligand, large, multinucleated, TRAP positive osteoclasts are seendemonstrating normal differentiation (panel b). The presence of thesiRNA specific for SEQ. ID. NO:1 in RAW cells resulted in a greatlyreduced ability of these cells to form large and mature osteoclasts inthe presence of RANK ligand (panels c-e). In addition to a decreasednumber of osteoclasts per well, the RAW-0440 cells were smaller and mostof exhibited a slight decrease in TRAP staining. Closer inspectionrevealed that these smaller osteoclasts were multinucleated suggestingnormal cellular fusion of the RAW-0440 precursors. Analysis of anotherRAW cell line, RAW-0440.mix, transfected with an equivalent amount ofall three siRNA expression vectors confirmed the previous phenotypicobservations. As before, the control cell line transfected with theempty vector formed large multinucleated osteoclasts that stained forTRAP (FIG. 4B, panel b). As shown in panel c, the RAW-0440.mixosteoclasts were multinucleated but small and fewer in number.

The effect of each siRNA was assessed by isolating total RNA from themature osteoclasts after 3 and 4 days of RANK ligand treatment andprobing with a fragment of the SEQ. ID. NO:1 cDNA to determine ifknockdown of endogenous gene expression occurred. A representativeexperiment is shown in FIG. 5. When 10 μg of total RNA was probed with afragment of the SEQ. ID. NO:1 cDNA, a single mRNA of 1.7 Kb wasobserved. A decrease, especially at day 4, in the amount of the SEQ. ID.NO1 mRNA was seen in the RNA isolated from the RAW-0440.1 cell lineindicating RNA interference occurred in these cells (FIG. 5, top panel,compare lanes 3 and 6). Similarly, the expression of two knownosteoclast marker genes, TRAP and Cathepsin K (Boyle et al., 2003), wassignificantly reduced in RAW-0440.1 cells (FIG. 5). Theosteoclast-specific character of SEQ. ID. NO:1 was evident by the lackof expression in the precursor cells (see lanes 1 and 4). The differencein the expression of the SEQ. ID. NO:1 gene was not due to thedifference in the amount of total RNA loaded on the gel as evidenced bythe probing of the same membrane with a fragment of the housekeepinggene glyceraldehyde-3-phosphate dehydrogenase (GAPDH, FIG. 5).

These results demonstrate that the absence of physiological levels ofSEQ. ID. NO:1 in RAW cells impairs their ability to differentiate intoosteoclasts properly and implies an important role for this gene inthese cells.

SEQ. ID. NO:2:

The sequences used for RNA interference were derived from thepolynucleotide SEQ. ID. NO:2 and have the SEQ. ID. NOs:67, 68 and 69.The cell lines derived from RAW cells transfected with plasmids encodingthe three siRNAs pd2-hU6/0179.1, pd2-hU6/0179.2 and, pd2-hU6/0179.3 arehereby-designated RAW-0179.1 (SEQ. ID. NO.:67), RAW-0179.2 (SEQ. ID.NO.:68), and RAW-0179.3 (SEQ. ID. NO.:69), respectively. In addition, asa positive control for normal osteoclastogenesis, RAW cells weretransfected with the empty vector (pd2-hU6) that does not contain anysiRNA. Phenotypic analysis of all cell lines is shown in FIG. 6. Panel aof FIG. 6 shows the control cell line, RAW-hU6, in the absence of RANKligand where the presence of multinucleated osteoclasts is not observedand the undifferentiated RAW cells are completely devoid of TRAPstaining. Upon treatment with RANK ligand, large and multinucleated,TRAP positive osteoclasts are seen demonstrating normal differentiation(panel b). Two of the siRNAs, namely those encoded by RAW-0179.1 andRAW-0179.2, resulted in a noticeable reduction in the ability of thesecells to form large, mature osteoclasts in the presence of RANK ligand(panels c,d). A third siRNA sequence, RAW-0179.3, was not effective (seeFIG. 6, panel e). In addition to a decreased number of osteoclasts perwell, the RAW-0179.1 and RAW-0179.2 cells were generally smaller andcontained less nuclei.

Isolating total RNA from the mature osteoclasts after 3 and 4 days ofRANK ligand treatment and probing with a fragment of the SEQ. ID. NO2cDNA to determine if knockdown of endogenous gene expression occurredassessed the effect of each siRNA. A representative experiment is shownin FIG. 7. When 10 μg of total RNA was probed with a fragment of theSEQ. ID. NO2 cDNA, two mRNAs of 2.3 Kb and 1.6 Kb were observed. Adecrease, especially at day 4, in the amount of the SEQ. ID. NO2 mRNAwas seen in the RNA isolated from the RAW-0179.1 cell line indicatingRNA interference occurred in these cells (FIG. 7, top panel, comparelanes 3 and 6). Furthermore, although the RAW-hU6 precursor cellsexpressed detectable levels of SEQ. ID. NO2 in the absence of RANKligand, expression was not seen in RNA from the RAW-0179.1 cells undersimilar conditions (compare lanes 1 and 4 in FIG. 7, top). Takentogether, these results show that effective RNA interference occurred.Two known osteoclast marker genes were also significantly reduced,especially Cathepsin K which was virtually undetectable in RAW-0179.1cells compared to the control cell line. The difference in theexpression of the SEQ. ID. NO2 gene was not due to the difference in theamount of total RNA loaded on the gel as evidenced by the probing of thesame membrane with a fragment of the housekeeping geneglyceraldehyde-3-phosphate dehydrogenase (GAPDH, FIG. 7).

These results demonstrate that SEQ. ID. NO2 is required for properosteoclast differentiation in RAW cells suggesting an important role forthis gene in these cells.

SEQ. ID. NO:3:

The sequences used for RNA interference were derived from thepolynucleotide SEQ. ID. NO:3 and have the SEQ. ID. NOs:70, 71 and 72.The use of RNA interference as described in the invention for SEQ. ID.NOs1 and 2 was applied to SEQ. ID. NO:3. The results obtained weresimilar showing that this gene is also required for properdifferentiation of RAW osteoclasts. An illustration of this result isdepicted in FIG. 8.

SEQ. ID. NO:4:

The sequences used for RNA interference were derived from thepolynucleotide SEQ. ID. NO:4 and have the SEQ. ID. NOs:73 and 74. Theuse of RNA interference as described in the invention for SEQ. ID. NOs1and 2 was applied to SEQ. ID. NO:4. The results obtained were similarshowing that this gene is required for proper differentiation of RAWosteoclasts. An illustration of this result is depicted in FIG. 9.

SEQ. ID. NO:5:

The sequences used for RNA interference were derived from thepolynucleotide SEQ. ID. NO:5 and have the SEQ. ID. NOs:75 and 76. Theuse of RNA interference as described in the invention for SEQ. ID. NOs1and 2 was applied to SEQ. ID. NO:5. The results obtained were similarshowing that this gene is required for proper differentiation of RAWosteoclasts. An illustration of this result is depicted in FIG. 10.

SEQ. ID. NO:6:

The sequences used for RNA interference were derived from thepolynucleotide SEQ. ID. NO:6 and have the SEQ. ID. NOs:77 and 78. Thesame approach for RNA interference was applied for this sequence withthe following modification. The siRNA expression plasmids pd2-hU6/1200.1and pd2-hU6/1200.2 were transfected as a mixture where equivalentamounts were used. As was observed for SEQ. ID. NO:1, pooling the siRNAexpression plasmids produces similar results to those obtained fromindividual plasmid transfections. Thus, the cell line that was obtainedfrom this transfection was termed RAW-1200.mix (see FIG. 11).

Treatment of this cell line with RANK ligand resulted in RAW cells thatdifferentiated sooner than the control cell line, RAW-hU6. In addition,the osteoclasts were larger and contained more nuclei per cell (comparepanels b and d in FIG. 11A). The experiment was repeated with TRAPstaining conducted at days 3, 4 and 5 five to directly compare theRAW-1200.mix line with the control. As shown in FIG. 11B, theosteoclasts from the RAW-1200.mix cells appeared much sooner and weremature by day 3, a point at which the control RAW cells are juststarting to form small multinucleated cells. Furthermore, osteoclastsderived from the RAW-1200.mix cell line seem to have a reduced survivalas a decrease in the number of remaining osteoclasts is observedstarting at day 4. The control cells are mature by day 4 and manyosteoclasts are still present even on day 5.

This result demonstrates that RNA interference of osteoclast-specificgenes using the approach of this invention not only identifies thosegenes that play a role in stimulating osteoclastogenesis, but alsoserves to validate those candidates that are negative regulators of thisprocess.

To further substantiate the observations described above, Northern blotanalysis was conducted on the total RNA isolated from the RAW-1220.mixcell line and compared to the control RAW-hU6. The blot was initiallyprobed with a fragment of the SEQ. ID. NO:6 cDNA but the message wasalmost undetectable by this method. The same blot was probed for theosteoclast marker genes, TRAP and Cathepsin K, as before. As shown inFIG. 12, the expression of TRAP was significantly increased in theRAW-1200.mix cells in agreement with the phenotypic observation.Cathepsin K was also upregulated albeit to a lesser extent. Again, GAPDHdemonstrated that equal amounts of RNA were loaded in each lane. Theseresults, like those from the osteoclastogenesis experiments, suggestthat SEQ. ID. NO:6 is a negative regulator of osteoclast differentiationin RAW cells.

SEQ. ID. NO:7:

The sequences used for RNA interference were derived from thepolynucleotide SEQ. ID. NO:7 and have the SEQ. ID. NOs:79 and 80. Theuse of RNA interference as described in the invention for SEQ. ID. NOs1and 2 was applied to SEQ. ID. NO:7. The results obtained (data notshown) were similar to those of SEQ. ID. NO:6 showing that knock-down ofthis gene resulted in an increase in osteoclast differentiationsuggesting that SEQ. ID. NO:7 is a negative regulator of this process inRAW cells.

SEQ. ID. NO:57:

The sequences used for RNA interference were derived from thepolynucleotide SEQ. ID. NO:57 and have the SEQ. ID. NOs:81 and 82. Theuse of RNA interference as described in the invention for SEQ. ID. NOs1and 2 was applied to SEQ. ID. NO:57. The results obtained were similarshowing that this gene is required for proper differentiation of RAWosteoclasts. An illustration of this result is depicted in FIG. 13.

L—Determination of Knockdown Effects on Bone Resorption

The functionality of the identified osteoclast-specific sequences wasexplored by seeding the cells on Osteologic™ (BD Biosciences,Mississauga, ON) discs to measure their bone resorptive activity.Osteologic™ discs are commercially available and contain a syntheticcalcium phosphate substrate and are well known to the skilled artisan asa model for bone degradation.

RAW cells were seeded in 24-well plates containing a calciumphosphate-coated disc (Osteologic™) at a density of 35 000 cells/well.Treatment with differentiation medium containing 100 ng/ml RANK ligandwas carried out for 5 days where after the osteoclasts were stained forTRAP expression as described above to determine the position and numberof multinucleated cells. Osteoclasts were removed with bleach andstained with 5% silver nitrate according to manufacturer's modified vonKossa method. Resorbed pits were observed microscopically. Thepercentage of resorbed surface area is determined by scanning thenegative image of the disc and using Photoshop™ (Adobe) to calculate thepercentage of black pixels at maximum contrast. The control, RAW-hU6,was set to the value of 1 and the maximal amount of resorption that wasobserved with the RAW-hU6 cell line in the presence of RANK ligand wasset to 100%.

SEQ. ID. NO:1:

In order to determine if the function of the RAW-0440 cell lines wereaffected by knockdown of SEQ. ID. NO:1, the cells were cultured anddifferentiated on Osteologic™ discs. An equal number of RAW cells wasseeded on each disc and treated with RANK ligand for a period of 4 daysbefore being fixed and stained by the manufacturer's modified von Kossamethod that stains the calcium phosphate substrate. White areas on thedisc indicate osteoclast resorption. As shown in FIG. 14A, the controlcell line, RAW-hU6, did not cause a large increase in the resorbed areaon the disc (panel a) but treatment of RANK ligand to induceosteoclastogenesis resulted in a significant amount of substrate beingdegraded by the osteoclasts (panel b). All three RAW-0440 cell lines hada reduced ability to degrade the substrate and the discs had a largerarea of unresorbed calcium phosphate substrate (panel c-e). The resultsare shown in FIG. 14B. The values (% black pixels) for the totalresorbed area for osteoclasts from the RAW-0440.1, RAW-0440.2, andRAW-0440.3 cell lines were 8.7%, 45.9%, and 22.2%, respectively. Theseresults indicate that targeting the SEQ. ID. NO:1 gene in osteoclastshas the effect of reducing their ability to resorb bone substrate.

SEQ. ID. NO:2:

The approach used for SEQ. ID. NO:1 was used to analyze the RAW-0179cell lines. As illustrated in FIG. 15, the osteoclasts exhibited areduced ability to resorb the substrate (FIG. 15A). Quantitativeanalysis of the remaining material on the discs showed that totalresorbed area was 36.1%, 29.4, and 51.2% for the RAW-0179.1, RAW-0179.2,and RAW-0179.3 cell lines, respectively (FIG. 15B). These resultsindicate that targeting the SEQ. ID. NO:2 gene in osteoclasts has theeffect of reducing their ability to resorb bone substrate.

M—Differential Expression of Human Orthologues of Some of the MurineOsteoclast-Specific Sequences:

The human orthologues for some of the murine osteoclast-specific geneshave been isolated using gene specific primers for RT-PCR amplificationand cloning of the corresponding double-stranded cDNA from mRNA of humanosteoclasts and their differential expression pattern measured using RNAfrom human CD34+ precursor and osteoclasts. Also, their tissuespecificity was determined using RNA from 30 different human tissues(adrenal, breast, jejunum, trachea, liver, placenta, aorta, brain, lung,adrenal cortex, esophagus, colon, ovary, kidney, prostate, thymus,skeletal muscle, vena cava, stomach, small intestine, heart, fallopiantube, spleen, bladder, cervix, pancreas, ileum, duodenum, thyroid andtesticule) purchased from Ambion (Austin, Tex.). All RNA samples wereamplified using RAMP and macroarrays were prepared as described inSection G. Each human orthologue cDNA was radiolabeled with α-³²P-dCTPusing a random priming procedure as specified by the supplier (Amersham,Piscataway, N.J.) and used as probe against the RNA present on themacroarrays. Hybridization and washing steps were performed followingstandard procedures well known in the art. FIG. 16A shows examples ofthe differential expression of human orthologues for SEQ. ID. NO:2 andSEQ. ID. NO:4 in the various tissues and osteoclasts samples representedon the macroarrays. However, in the case of SEQ. ID. NO:1, thehybridization signal on the macroarray was undetectable due to therelatively low abundance of this sequence. As such, standard RT-PCR withgene specific primers for human TSP50 was performed on human precursorand osteoclast samples in order to measure its expression (FIG. 16B).The 1.3 Kb TSP50 PCR amplicon was detected in the human osteoclastsamples for both donor 1 and donor 4 (FIG. 16B, Lanes 2 and 4respectively) but not the corresponding precursor samples (FIG. 16B,Lanes 1 and 3 respectively). The 1.3 Kb PCR amplicon was confirmed bysequence analysis as TSP50. It is evident from these results that thehuman orthologues of some of the murine selected osteoclast-specificsequences are similarly upregulated in the human CD34+ derivedosteoclasts and are also highly specific. Thus, these results suggestthat the use of the murine RAW264.7 model to identifyosteoclast-specific genes involved in human osteoclastogenesis is avalid strategy.

N—Biological Validation of the Human Orthologue for SEQ. ID, NO. 1 (SEQ.ID. NO, 88) in Osteoclastogenesis.

In order to validate the biological significance of the human orthologuefor SEQ. ID. NO. 1 (SEQ. ID. NO.88), it was important to demonstratethat the function observed in the mouse osteoclast model for SEQ. ID.NO. 1 was conserved in human osteoclasts. Unlike the mouse model where acell line could be used for osteoclast differentiation, no equivalentmodel exists in humans. Thus, validation studies were conducted in humanprimary bone marrow cells using a commercial lentiviral short hairpin(sh) RNA delivery system as described by the manufacturer (Invitrogen,Burlington, ON) unless otherwise stated. The siRNA sequence,5′-CTGCCTGATCTGGCGTGAT-3′ (SEQ ID. NO. 90) was used to specificallytarget SEQ. ID. NO. 88, the coding sequence of which was cloned from ahuman osteoclast cDNA library in-house.

A template for the expression of the shRNA was cloned into thelentiviral expression vector and co-transfected in 293FT cells withexpression vectors for the viral structural proteins. After two days,supernatants containing the lentivirus were collected and stored at −80°C. After titering, 20 MOIs (multiplicity of infection) were used toinfect human osteoclast precursors purchased from Cambrex (EastRutherford, N.J.). The following day, the medium was replaced with freshmedium containing RANK ligand to initiate osteoclast differentiation.Approximately 7 days later, the cells were fixed and TRAP stainingperformed as described in section B—Preparation of osteoclastdifferentiated cells. In parallel, lentiviral particles containing acontrol shRNA against β-galactosidase were also used to infect the humanosteoclast precursor cells.

FIG. 17 shows that infection of human bone marrow cells withlentiviruses expressing the specific shRNA for SEQ. ID. NO. 88 (AB0440siRNA) resulted in a marked decrease of TRAP-positive multinucleatedosteoclasts compared to human bone marrow cells infected withlentiviruses expressing the control shRNA (control siRNA) (see arrows inleft panel of FIG. 17) in the presence of RANK ligand. These resultswere in agreement with the validation results obtained in the mousemodel (section K-Results of RNA Interference studies) and thus, evidencethat the human orthologue for SEQ. ID. NO. 1 (SEQ. ID. NO. 88) plays asimilarly important role in differentiation of human osteoclasts.

O—A Functional Complementation Assay for SEQ. ID. NO. 88 to Screen forInhibitors of Osteoclastogenesis.

A complementation assay was developed to test the function of SEQ. ID.NO. 88 in the differentiation of mouse osteoclasts from RAW264.7 cellsdevoid of the corresponding endogenous mouse protein. The RAW264.7 cellline containing the mouse-specific shRNA (RAW-AB0440si) for SEQ. ID. NO.1, which showed greatly reduced ability to differentiate into matureosteoclasts, was transfected with an eukaryotic expression vectorcontaining the entire coding sequence for SEQ. ID. NO. 88, termedlp200-hAB0440. This lp200 expression vector (SEQ. ID. NO. 91) wasmodified from a commercial vector, pd2-EGFP-N1 (Clontech, Mountain View,Calif.) where the NEO-KAN antibiotic cassette was replaced by ahygromycin resistance gene for selection in mammalian cells and anampicillin resistance gene for propagation in prokaryotes. Expression ofthe inserted human gene sequence is under control of a strong CMVpromoter in lp200. Approximately 2.5×10⁵ RAW-0440si cells/well wereseeded in 6-well plates and transfected with either 1 μg lp200 orlp200-hAB0440 using Fugene 6 (Roche, Laval, QC), and stabletransfectants selected for 5 days in the presence of 50 μg/mlhygromycin. Two RAW 264.7-0440si stable cell lines were selected—onethat expressed SEQ. ID. NO. 88 (lp200-hAB0440) and the other containingonly the vector (lp200). After expansion of these two cell lines, 4 000cells/well for each were seeded in 96-well plates and left eitheruntreated or treated for 4 days with 100 ng/ml RANK ligand. The cellswere fixed and stained for TRAP expression in order to visualize matureosteoclasts.

FIG. 18 shows that the RAW-0440si cells transfected with only the emptylp200 vector were unable to efficiently form osteoclasts (left panels).Conversely, the cells transfected with lp200-hAB0440 (SEQ. ID. NO. 88)were rescued (complemented) and thus, differentiated in response to RANKligand treatment into osteoclasts (right panels). These results confirmthat the function for the mouse and human sequences corresponding toSEQ. ID. NO. 1 is conserved and essential for osteoclastdifferentiation.

Thus, it is anticipated that this type of complementation cell-basedassay may serve as the basis for screening compounds capable of bindingto and inhibiting the function of SEQ. ID. NO. 88. A compound librarymay be applied to this ‘rescued’ cell line in order to identifymolecules (small molecule drugs, peptides, or antibodies) capable ofinhibiting the complementation effect of SEQ. ID. NO. 88. Consequently,any measurable reduction in osteoclast differentiation would beindicative of compounds that attenuate the complementation activity ofSEQ. ID. NO. 88 in the assay. It is further anticipated that this assayformat may be applicable to any gene required for differentiation ofRAW264.7 cells into osteoclast, which may be used for drug screening.

P—The Human Orthologue Protein of SEQ. ID. NO. 9 (SEQ. ID. NO. 88) isMembrane-Bound and Glycosylated.

It is contemplated in the literature that SEQ. ID. NO. 88 may encode amembrane-bound or secreted protease, termed Tsp50. In order to determineif the polypeptide for SEQ. ID. NO. 88 is truly membrane-bound orsecreted, a plasmid (pCMX-HA-hAB0440) containing the entire codingsequence for SEQ. ID. NO. 88 was constructed. The expression vector,pCMX-HA (SEQ. ID. NO. 92) contains a strong

CMV promoter for expression of the HA epitope and cDNA insert.Approximately, 2.5×10⁵ Cos-7 cells/well were seeded in 6-well plates andtransiently transfected with 1 μg of the pCMX-HA-hAB0440 expressionplasmid using Fugene 6 (Roche, Laval, QC). Cells in some wells were nottreated (−) while others were treated with either 2 μg/ml tunicamycin(T) for 24 hours or 0.5 units/ml phosphoinositol phospholipase C (P) for1 hour. Tunicamycin blocks the reaction of UDP-GlcNAc and Dol-P in thefirst step of glycoprotein synthesis, thus inhibiting the synthesis ofall N-linked glycoproteins. Phosphoinositol phospholipase C specificallycleaves glycosyl-phosphoinositol (GPI) linkages, which releases GPIanchored proteins into the surrounding medium. The expressed HA fusedpolypeptide for SEQ. ID. NO. 88 was detected by Western blot analysiswith an anti-HA antibody (Sigma, St. Louis, Mo.). Following lysis of thepCMX-HA-hAB0440-Cos-7 cells, soluble fractions were prepared, separatedon a SDS-polyacrylamide gel and transferred to a PVDF membrane. Theprotein blot was then incubated with anti-HA antibody for 1 hour and thebands visualized using the ECL kit from Roche (Laval, QC). The sameWestern blot was stripped and reacted with an anti-actin antibody tocontrol for equal loading of protein samples.

FIG. 19 shows a polypeptide with a predicted size of 37 KDacorresponding to the full-length polypeptide for SEQ. ID. NO. 88.Interestingly, expression of this polypeptide was only observed when thetransiently transfected cells were treated with tunicamycin in thepresence of 10% FBS in the culture media compared to serum-starved cells(0% FBS) (FIG. 19, Lanes T). This finding suggested that inhibition ofN-linked glycosylation resulted in trapping of the SEQ. ID. NO. 88polypeptide within the cells, which is evidence that the protein isglycosylated. Following treatment with phosphoinositol phospholipase C,the SEQ. ID. NO. 88 polypeptide was no longer detected in the solublefraction (FIG. 19, Lanes P), which suggested that it was released intothe media likely due to cleavage of the proposed GPI linkage. As acontrol for equal loading, the membrane was stripped and reacted with anantibody against the housekeeping protein, α-actin, which showed thatthe observed differences in expression of the SEQ. ID. NO. 88polypeptide was not a result of unequal loading of the gel (FIG. 19,α-actin panel).

Q—Inhibition of RAW264.7 Differentiation into Osteoclast Using aMonoclonal Anti-Tsp50 (SEQ. ID. NO. 1) Antibody.

In light of the results demonstrating that of the polypeptide for SEQ.ID. NO. 88 (depicted in SEQ ID NO.:153) is essential for osteoclastdifferentiation (see Example N) and it is localized at the cell surface(see Example P), then this protein represents an excellent candidate forthe development of an antibody therapeutic strategy for treating thesymptoms of osteoporosis. A monoclonal antibody against mouse Tsp50 (R&DSystems, Minneapolis, Minn.) was purchased and used to test whether ornot a specific antibody against the polypeptide for SEQ. ID. NO. 1(depicted in SEQ ID NO.:93) would inhibit osteoclast differentiation inthe RAW 264.7 model. Approximately, 4 000 RAW264.7 cells/well wereseeded in 96-well plates and treated with 100 ng/ml RANK ligand in thepresence of increasing concentrations of either the mouse monoclonalantibody against Tsp50 or a control anti-HA antibody (Sigma, St. Louis,Mo.). Three days later, the cells were fixed and stained for TRAPexpression and the multinucleated cells were scored.

FIG. 20A is a histogram showing that increasing concentrations ofanti-Tsp50 antibody (anti-AB0440) resulted in a dose-dependent decreasein the number of multinucleated osteoclasts with maximal inhibition seenat 50 μg/ml. Whereas, treatment of the RAW264.7 cells with equivalentconcentrations of the anti-HA antibody resulted in no statisticallysignificant effect. FIG. 20A represents an average of two experimentsconducted in triplicate. Treatment with the anti-AB0440 did not resultin death of the RAW264.7 cells but rather, inhibition of differentiationas measured by the loss in mature osteoclasts seen after TRAP stainingand no significant reduction in precursor cell numbers (FIG. 20B). Theseresults indicate that antibodies which specifically targetosteoclast-specific cell surface or secreted proteins required fordifferentiation, as exemplified by anti-AB0440, have the potential toserve as therapeutic drugs for treating osteoporosis by reducingosteoclast numbers and consequently, bone resorption activity. It iscontemplated that recombinant and/or monoclonal antibodies developed tothe polypeptide for SEQ. ID. NO. 88 may function similarly toanti-AB0440 seen for the mouse model in this example.

R—Development of a Functional Interaction Assay to Screen for Inhibitorsof Osteoclast Activity Using SEQ. ID. NO. 2 as a Model.

SEQ. ID. NO. 2 (AB0179) belongs to an osteoclast-specific vacuolar(v)-ATPase, a large protein complex containing several subunits. Thev-ATPase a subunit comprises four isoforms (a1-a4), which constitutesthe V₀ domain. This domain is important for the hydrolysis of ATP inorder to provide the energy required for the secretion of protons acrossthe plasma membrane into the pocket that is created between the ruffledmembrane of the mature osteoclast and the bone surface. In osteoclasts,the a3 subunit interacts with the d subunit, which is important for thestructural integrity of the ATPase complex (Nishi and Forgac, 2002).There are two d subunits in humans, d1 and d2, the latter found almostexclusively in osteoclasts and is coded for by the human orthologue ofSEQ. ID. NO. 2 which corresponds to polynucleotide SEQ. ID. NO. 89(encoding SEQ ID NO.:154). Validation studies using the RAW264.7 modelhave clearly demonstrated the importance of d2 in osteoclast function(section K—Results of RNA Interference studies) where in the presence ofsiRNAs against SEQ. ID. NO. 2, the bone resorbing activity of matureosteoclasts was markedly reduced. Additionally, it has been welldocumented that the a3 isoform is the major form of the v-ATPase asubunit in osteoclasts and bone in general (Smith et al., 2005). Thus,in order to identify molecules that are capable of inhibiting thefunction of the v-ATPase in osteoclasts, the specific interactionbetween the d2 and a3 subunits will be exploited.

The expression of the d isoforms in human osteoclast and precursor cells(HOPs) was measured by Northern blot analysis. Approximately, 1.5×10⁴precursor cells/well were seeded in 96-well plates and a portion wastreated with 33 μg/ml M-CSF and 100 ng/ml RANK ligand for 7 days to formosteoclasts. Total RNA was prepared from the precursors and osteoclastsusing Trizol™ (Invitrogen, Burlington, ON) and 10 μg/lane waselectrophoresed in a 1% agarose/TAE gel. The RNA was electrotransferedto a nylon membrane and hybridized sequentially to [³²P]dCTP labeledprobes specific for the d2 (SEQ. ID. NO. 89) and d1 subunits, for theosteoclast-specific gene, Cathepsin K and for the housekeeping gene,β-actin. The washed membrane was exposed to film for the required amountof time to detect the corresponding mRNA bands.

FIG. 21A shows that SEQ. ID. NO. 89 (d2) was upregulated in response toRANK ligand (Panel 1; Lane RL) compared to precursors (Panel 1; Lane -).With the probe specific for v-ATPase d1, the opposite expression patternwas observed indicating that this d isoform was downregulated inosteoclasts (Panel 2; Lane RL) compared to precursors (Panel 2; Lane -).As expected, the osteoclast-specific gene, Cathepsin K was highlyupregulated in response to RANK ligand and not present in the precursors(Panel 3). Equal loading of the RNA samples was evident by thenon-differential expression pattern of the housekeeping gene, β-actin(Panel 4). Since a3 is predominantly found in the v-ATPase a subunit ofosteoclasts, these results then suggests that the d2 subunit would mostlikely be complexed with the a3 subunit in human osteoclasts v-ATPase.Thus, isolation of a specific inhibitor of this interaction wouldpreferentially reduce the osteoclast-specific v-ATPase activity andthus, bone resorption.

In order to experimentally demonstrate the interaction between d2 anda3, the coding sequence corresponding to SEQ. ID. NO. 89 (d2) (SEQ IDNO.:154) was cloned into the prokaryotic expression vector, pGEX-2T(Pharmacia, GE Healthcare), expressed as a GST fusion protein in E. coliand purified with glutathione beads. In parallel, cDNA fragments ofmouse v-ATPase-a3 (amino acids 1-385) and v-ATPase a4 (amino acids1-388) were cloned into the eukaryotic expression vector, pCMX-Flag inorder to express a Flag-tagged a3 subunit or a4 subunit in mammaliancells. The pCMX-Flag/v-ATPase-a3 and pCMX-Flag/v-ATPase-a4 recombinantplasmids were transfected in 293FT cells and cell lysates were generatedin which, the v-ATPase-a3 and v-ATPase-a4 FLAG-tagged polypeptides werereadily detected with an anti-Flag antibody (FIG. 21B, II, upper panel).

In order to measure the interaction between d2 (SEQ. ID. NO. 89) and a3or a4, equal amounts of 293FT lysates containing either Flag-tagged a3or a4 were incubated with purified GST or GST-d2 at 4° C. for 90 minutesunder mild agitation. After washing, the protein mixes were separated ona SDS-PAGE and transferred to PVDF membrane. The membrane was thenincubated with anti-Flag antibody (Sigma, St. Louis, Mo.) and the bandsvisualized using the ECL kit from Roche (Laval, QC). Clearly, only thea3 fragment could be detected in the GST-d2 reactions compared to theGST reactions indicating a specific interaction between d2 and a3 (FIG.21B, I-a3, upper panel) but not between d2 and a4 (FIG. 21B, I-a4, upperpanel). The membrane was then re-probed with anti-GST antibody, whichshowed that equal amounts of GST fusion protein were used in eachreaction (FIG. 21B, I, lower panel). Additionally, the use of ananti-Flag antibody showed that equal quantities of a3 and a4 werepresent in the binding reactions (FIG. 21B, II, upper panel) and thesame membrane re-probed with an anti-actin antibody, demonstrated thatequal amounts of the corresponding cell lysate was used (FIG. 21B, II,lower panel). Thus, this observed specific interaction between d2 and a3forms the basis for developing a screening assay to interrogate compoundlibraries (small molecule drugs, peptides, or antibodies) in order toidentify those compounds capable of inhibiting this interaction. Such ascreening assay may be developed as a FRET (fluorescence resonanceenergy transfer) method or any similar methods, which are highlysensitive and easily upscalable for high throughput screening inmultiwell plates. These compounds may be useful as therapeutics formodulating the bone resorption activity of osteoclasts by inhibiting thefunction of the osteoclast-specific v-ATPases.

v-ATPases present in other tissues, most notably the kidney (Nishi andForgac, 2002) where the d2 gene is expressed at low levels as shown byus and others (Nishi et al., 2003; Smith et al., 2005) do not appear tocontain the a3 subunit but rather, the a4 subunit (Stehberger et al.,2003; Smith et al., 2000). Therefore, an inhibitor of the interactionbetween d2 and a3 would preferentially be effective in human osteoclastsand would not interfere with v-ATPases in other tissues.

One of skill in the art will readily recognize that orthologues for allmammals maybe identified and verified using well-established techniquesin the art, and that this disclosure is in no way limited to one mammal.The term “mammal(s)” for purposes of this disclosure refers to anyanimal classified as a mammal, including humans, domestic and farmanimals, and zoo, sports, or pet animals, such as dogs, cats, cattle,horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal ishuman.

The sequences in the experiments discussed above are representative ofthe NSEQ being claimed and in no way limit the scope of the invention.The disclosure of the roles of the NSEQs in osteoclastogenesis andosteoclast function satisfies a need in the art to better understand thebone remodeling process, providing new compositions that are useful forthe diagnosis, prognosis, treatment, prevention and evaluation oftherapies for bone remodeling and associated disorders.

The art of genetic manipulation, molecular biology and pharmaceuticaltarget development have advanced considerably in the last two decades.It will be readily apparent to those skilled in the art that newlyidentified functions for genetic sequences and corresponding proteinsequences allows those sequences, variants and derivatives to be useddirectly or indirectly in real world applications for the development ofresearch tools, diagnostic tools, therapies and treatments for disordersor disease states in which the genetic sequences have been implicated.

Although the present invention has been described hereinabove by way ofpreferred embodiments thereof, it maybe modified, without departing fromthe spirit and nature of the subject invention as defined in theappended claims.

TABLE 1 Differentially expressed sequences found in osteoclasts anddemonstrated to have an effect on osteoclastogenesis followinginhibition with specific siRNAs. NCBI ORF Unigene Nucleotide Nucleotide#/Gene Positions/ Sequence Symbol/Gene Accession Polypeptide No. IDNumber sequence No. Function SEQ ID NO. 1 Mm.102265/ NM_146227 26-1345peptidase activity Tsp50/ encoding 235631 SEQ IDNO.: 93 SEQ ID NO. 2Mm.19298/ NM_175406 70-1122 hydrogen-transporting Atp6v0d2/ encoding SEQATPase activity, 242341 ID NO.: 94 rotational mechanism SEQ ID NO. 3Mm.20904/ NM_019922 72-1274 extracellular space Crtap/ encoding protein;function 56693 SEQ ID NO.: unknown 95 SEQ ID NO. 4 Mm.12654/ NM_175474314-1114 hypothetical protein A230106M15Rik/ encoding SEQ LOC231717;function 231717 ID NO.: 96 unknown SEQ ID NO. 5 Mm.181860/ NM_02647328-1371 GTPase activity; Tubb6/ encoding SEQ structural molecule 67951ID NO.: 97 activity SEQ ID NO. 6 Mm.323393/ NM_028792 905-1513hypothetical protein Josd1/ encoding SEQ LOC74158; function 74158 IDNO.: 98 unknown SEQ ID NO. 7 Mm.332739/ NM_022964 162-737 extracellularspace; Lat2/ encoding SEQ function unknown 56743 ID NO.: 99 SEQ ID NO.Mm.103560/ NM_030887 232-723 transcriptional repressor 57 Jundm2/encoding SEQ activity; shown to play a 81703 ID NO.: 100 role inRANK-mediated signal transduction, especially in osteoclastdifferentiation

TABLE 2 Differentially expressed sequences found in osteoclasts withputative roles in bone remodeling. NCBI ORF Unigene NucleotideNucleotide #/Gene Positions/ Sequence Symbol/Gene Accession PolypeptideNo. ID Number sequence No. Function SEQ ID NO. 8 Mm.10154/ NM_0115711497-3380 Protein kinase activity Tesk1/ encoding SEQ 21754 ID NO.: 101SEQ ID NO. 9 Mm.142827/ NM_138604 287-1987 hypothetical protein Otud5/encoding SEQ LOC54644 54644 ID NO.: 102 SEQ ID NO. Mm.146001/ BC0046781-311 Ras association 10 Rassf8/71323 encoding SEQ possibly involved inID NO.: 103 signal transduction SEQ ID NO. Mm.153014/ BC068244 251-1246possibly involved in 11 Gcn1l1/ encoding SEQ amino acid 231659 ID NO.:104 biosynthesis with exact function unknown SEQ ID NO. Mm.153159/NM_009838 55-1650 chaperonin containing 12 Cct6a/ encoding SEQ TCP-1possibly 12466 ID NO.: 105 involved in protein folding and binding SEQID NO. Mm.157103/ NM_133943 53-487 involved in steroid 13 Hsd3b7/encoding SEQ biosynthesis 101502 ID NO.: 106 SEQ ID NO. Mm.169234/NM_026452 6-947 hypothetical protein 14 2310005O14Rik/ encoding SEQLOC67914 67914 ID NO.: 107 SEQ ID NO. Mm.266341/ NM_010939 537-3281receptor activity and 15 Nrp2/ encoding SEQ cell adhesion 18187 ID NO.:108 SEQ ID NO. Mm.17917/ NM_025994 55-777 calcium ion binding 16 Efhd2/encoding SEQ 27984 ID NO.: 109 SEQ ID NO. Mm.200499/ NM_146165 116-958protein kinase or 17 Eif2ak1/ encoding SEQ transferase activity 15467 IDNO.: 110 SEQ ID NO. Mm.20845/ NM_013790 200-4510 ATPase activity, 18Abcc5/ encoding SEQ coupled to 27416 ID NO.: 111 transmembrane movementof substances SEQ ID NO. Mm.21880/ NM_019752 146-1522 proteolysis 19Htra2/ encoding SEQ 64704 ID NO.: 112 SEQ ID NO. Mm.2271/ NM_011338154-522 chemokine activity 20 Ccl9/ encoding SEQ 20308 ID NO.: 113 SEQID NO. Mm.24684/ NM_008037 171-1151 regulation of 21 Fosl2/ encoding SEQtranscription, DNA- 14284 ID NO.: 114 dependent SEQ ID NO. Mm.251199/NM_025549 139-1029 molecular function 22 Arrdc4/ encoding SEQ unknown66412 ID NO.: 115 SEQ ID NO. Mm.2534/ NM_011193 242-1489 cell adhesion23 Pstpip1/ encoding SEQ 19200 ID NO.: 116 SEQ ID NO. Mm.266592/XM_488832 1-987 molecular function 24 C030034I22Rik/ encoding SEQunknown 77533 ID NO.: 117 SEQ ID NO. Mm.268165/ BC029223 475-1920caspase activity 25 Cflar/ encoding SEQ 12633 ID NO.: 118 SEQ ID NO.Mm.272047/ BC060114 246-1286 helicase activity 26 Helz/ encoding SEQ78455 ID NO.: 119 SEQ ID NO. Mm.278726/ NM_028108 296-802N-acetyltransferase 27 Mak3/ encoding SEQ activity 72117 ID NO.: 120 SEQID NO. Mm.279861/ BC024419 49-432 regulation of 28 Polr2f/ encoding SEQtranscription 69833 ID NO.: 121 SEQ ID NO. Mm.280895/ BC023789/ 218-1003kinase activity/ 29 Uck2; AI481316 encoding SEQ hypothetical proteinAI481316/ ID NO.: 122 LOC98383 80914; 98383 SEQ ID NO. Mm.217216/BC095943 574-3920 intracellular signaling 30 Magi1/ encoding SEQ cascade14924 ID NO.: 123 SEQ ID NO. Mm.286536/ NM_133754 137-1264 cell adhesion31 Fblim1/ encoding SEQ 74202 ID NO.: 124 SEQ ID NO. Mm.286753/NM_019492 236-2068 signal transducer 32 Rgs3/ encoding SEQ activity50780 ID NO.: 125 SEQ ID NO. Mm.298728/ NM_022656 335-4399 receptoractivity; 33 Nisch/ encoding SEQ integrin binding 64652 ID NO.: 126 SEQID NO. Mm.129840/ BC050783 371-3727 hypothetical protein 349430063L05Rik/ encoding SEQ LOC229622; function 229622 ID NO.: 127unknown SEQ ID NO. Mm.31672/ NM_009873 56-1036 cyclin-dependent 35 Cdk6/encoding SEQ protein kinase activity 12571 ID NO.: 128 SEQ ID NO.Mm.331198/ BC057030 94-1776 transcription factor 36 Tdrkh/ encoding SEQ72634 ID NO.: 129 SEQ ID NO. Mm.44901/ NM_026620 30-1319 functionunknown 37 2610510H03Rik/ encoding SEQ 68215 ID NO.: 130 SEQ ID NO.Mm.348047/ BC046620 329-610 function unknown 38 Usmg4/ encoding SEQ83679 ID NO.: 131 SEQ ID NO. Mm.37803/ BC030438 No significant 39Specc1/ predicted ORF at 432572 present/ function unknown SEQ ID NO.Mm.295306/ BC025076 123-494 hypothetical protein 40 BC025076/ encodingSEQ LOC216829; function 216829 ID NO.: 132 unknown SEQ ID NO. Mm.45815/BC023930 405-2867 guanyl-nucleotide 41 Bcar3/ encoding SEQ exchangefactor 29815 ID NO.: 133 activity SEQ ID NO. Mm.4615/ NM_016747 325-2874function unknown 42 Dlgh3/ encoding SEQ 53310 ID NO.: 134 SEQ ID NO.Mm.86572/ BC017612 192-371 function unknown 43 BC017612/ encoding SEQ170748 ID NO.: 135 SEQ ID NO. Mm.354736/ XM_137276 730-2781 functionunknown 44 Gas2l3/ encoding SEQ 237436 ID NO.: 136 SEQ ID NO. Mm.240265/BC027051 81-428 hypothetical protein 45 5830415L20Rik/ encoding SEQLOC68152; function 68152 ID NO.: 137 unknown SEQ ID NO. Mm.27545/BC002249 25-1056 S- 46 Hrmt1l2/ encoding SEQ adenosylmethionine- 15469ID NO.: 138 dependent methyltransferase activity SEQ ID NO. Mm.28071/BC027508 65-358 integral to membrane; 47 1810030N24Rik/ encoding SEQfunction unknown 66291 ID NO.: 139 SEQ ID NO. Mm.293761/ BC04629556-1237 transferase activity, 48 Pofut1/ encoding SEQ transferringglycosyl 140484 ID NO.: 140 groups SEQ ID NO. Mm.341204/ NM_011365258-5234 calcium ion binding 49 Itsn2/ encoding SEQ 20403 ID NO.: 141SEQ ID NO. Mm.347964/ BC026949 189-872 hydrolase activity; 50 Fahd1/encoding SEQ calcium ion binding 68636 ID NO.: 142 activity SEQ ID NO.Mm.46513/ NM_025998 87-710 hypothetical protein 51 2610200G18Rik/encoding SEQ LOC67149; integral to 67149 ID NO.: 143 membrane; functionunknown SEQ ID NO. Mm.6743/ NM_008686 249-2474 regulation of 52 Nfe2l1/encoding SEQ transcription, DNA- 18023 ID NO.: 144 dependent SEQ ID NO.Mm.78861/ NM_053086 30-1229 nucleolus organization 53 Nolc1/ encodingSEQ and biogenesis 70769 ID NO.: 145 SEQ ID NO. Mm.86437/ BC05481759-601 hydrolase activity; 54 Spcs3/ encoding SEQ receptor activity76687 ID NO.: 146 SEQ ID NO. Mm.296902/ BC017613 264-1619 functionunknown 55 Tapbpl/ encoding SEQ 213233 ID NO.: 147 SEQ ID NO. Mm.159563/XM_128030 50-1294 transmembrane 7 56 Tm7sf4/ encoding SEQ superfamilymember 4; 75766 ID NO.: 148 function unknown

TABLE 3 List of mRNA spliced variants for some Sequence IDs isolatedthus far from mouse and human osteoclast RNA samples Nucleotide ORFSequence Spliced Variant Nucleotide Polypeptide No. IdentificationPositions sequence No. SEQ ID NO. 0440-TO4-22-mFL_15 21-1259 SEQ ID NO.:149 83 SEQ ID NO. 0179-SL22-hFL_36 40-1092 SEQ ID NO.: 150 84 (humanorthologue variant #1) SEQ ID NO. 0179-SL22-hFL_1 40-978  SEQ ID NO.:155 85 (human orthologue variant #2) SEQ ID NO. 0799-SL22- 40-978  SEQID NO.: 151 86 mFL_3_(TAG1- 3′UTR) SEQ ID NO. 0799-SL22- 124-750  SEQ IDNO.: 152 87 mFL_4_(TAG1- 3′UTR)

TABLE 4 List of some human orthologue NCBI ORF Sequence UnigeneAccession Nucleotide Polypeptide Identification Cluster Number Positionssequence No. SEQ ID NO. 88 Hs.120365 NM_013270 51-1208 SEQ ID NO.: 153SEQ ID NO. 89 Hs.436360 NM_152565 70-1122 SEQ ID NO.: 154

TABLE 5 list of additional sequences identification of plasmids andsiRNA oligonucleotides Sequence Identification name Description SEQ. ID.NO. 58 p14 Vector for STAR SEQ. ID. NO. 59 p17+ Vector for STAR SEQ. ID.NO. 60 pCATRMAN Vector for STAR SEQ. ID. NO. 61 p20 Vector for STAR SEQ.ID. NO. 62 OGS 77 Primer used for STAR p14 vector SEQ. ID. NO. 63 OGS302 Primer used for STAR p17+ vector SEQ. ID. NO: 64 0440.1 siRNAsequence for SEQ. ID. NO. 1 SEQ. ID. NO: 65 0440.2 siRNA sequence forSEQ. ID. NO. 1 SEQ. ID. NO: 66 0440.3 siRNA sequence for SEQ. ID. NO. 1SEQ. ID. NO: 67 0.179.1 siRNA sequence for SEQ ID NO.: 2 SEQ. ID. NO: 680.179.2 siRNA sequence for SEQ ID NO.: 2 SEQ. ID. NO: 69 0.179.3 siRNAsequence for SEQ ID NO.: 2 SEQ. ID. NO: 70 0799.1 siRNA sequence for SEQID NO.: 3 SEQ. ID. NO: 71 0799.2 siRNA sequence for SEQ ID NO.: 3 SEQ.ID. NO: 72 0799.3 siRNA sequence for SEQ ID NO.: 3 SEQ. ID. NO: 730351.1 siRNA sequence for SEQ ID NO.: 4 SEQ. ID. NO: 74 0351.2 siRNAsequence for SEQ ID NO.: 4 SEQ. ID. NO: 75 1035.1 siRNA sequence for SEQID NO.: 5 SEQ. ID. NO: 76 1035.2 siRNA sequence for SEQ ID NO.: 5 SEQ.ID. NO: 77 1200.1 siRNA sequence for SEQ ID NO.: 6 SEQ. ID. NO: 781200.2 siRNA sequence for SEQ ID NO.: 6 SEQ. ID. NO: 79 0233A.1   siRNAsequence for SEQ ID NO.: 7 SEQ. ID. NO: 80 0233A.2   siRNA sequence forSEQ ID NO.: 7 SEQ. ID. NO: 81 0682.1 siRNA sequence for SEQ ID NO.: 57SEQ. ID. NO: 82 0682.2 siRNA sequence for SEQ ID NO.: 57 SEQ. ID. NO. 90siRNA sequence for SEQ. ID. NO. 88 SEQ. ID. NO. 91 Ip200 expressionvector SEQ. ID. NO. 92 pCMX-HA expression vector

REFERENCES Patents

U.S. Pat. No. 5,712,127 Malek et al., Jan. 27, 1998

U.S. Pat. No. 6,498,024, Malek et al., Dec. 24, 2002

(U.S. patent application Ser. No. 11/000,958 field on Dec. 2, 2003published under No. US 2005/0153333A1 on Jul. 14, 2005 and entitled“Selective Terminal Tagging of Nucleic Acids”

U.S. Pat. No. 6,617,434 Duffy, Sep. 9, 2003

U.S. Pat. No. 6,451,555 Duffy, Sep. 17, 2002

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1-55. (canceled)
 56. A method for inhibiting bone resorption comprisingadministering to a subject in need an antibody or an antigen bindingfragment capable of specific binding to a polypeptide having at least67% sequence similarity with SEQ ID NO.:153.
 57. The method of claim 56,wherein the polypeptide is capable of inducing differentiation ofosteoclasts.
 58. The method of claim 56, wherein the polypeptide has asequence at least 80% identical, at least 90% identical or at least 95%identical to SEQ ID NO.:153 or SEQ ID NO.:93.
 59. The method of claim56, wherein the polypeptide has a sequence identical to SEQ ID NO.:153or SEQ ID NO.:93.
 60. The method of claim 56, wherein the antibody orantigen binding fragment is capable of impairing an activity of thepolypeptide in osteoclast precursor cells or in osteoclasts.
 61. Themethod of claim 56, wherein the antibody or antigen binding fragmentinhibits osteoclast differentiation.
 62. The method of claim 56, whereinthe antibody is selected from the group consisting of a polyclonalantibody, a monoclonal antibody, a chimeric antibody, a humanizedantibody and a human antibody.
 63. The method of claim 56, wherein theantigen binding fragment is a FV, a Fab, a Fab′ or a (Fab′)₂.
 64. Themethod of claim 56, wherein the antibody or antigen binding fragment isadministered in combination with a drug or an hormone.
 65. The method ofclaim 64, wherein the drug is an antiresorptive drug or a drugincreasing bone mineral density.
 66. The method of claim 56, wherein theantibody or antigen binding fragment is conjugated with a therapeuticagent.
 67. The method of claim 56, wherein the subject in need suffersfrom a bone remodelling disorder.
 68. The method of claim 67, whereinthe bone remodelling disorder is associated with a decrease in bonemass.
 69. The method of claim 67, wherein the bone remodelling disorderis selected from the group consisting of osteoporosis, osteopenia,osteomalacia, hyperparathyroidism, hyperthyroidism, hypogonadism,thyrotoxicosis, systemic mastocytosis, adult hypophosphatasia,hyperadrenocorticism, osteogenesis imperfecta, Paget's disease,Cushing's disease/syndrome, Tumer syndrome, Gaucher disease,Ehlers-Danlos syndrome, Marfan's syndrome, Menkes' syndrome, Fanconi'ssyndrome, multiple myeloma, hypercalcemia, hypocalcemia, arthritides,periodontal disease, rickets, fibrogenesis imperfecta ossium,osteosclerotic disorders such as pycnodysostosis and damage caused bymacrophage-mediated inflammatory processes.
 70. A pharmaceuticalcomposition comprising: a. An antibody or an antigen binding fragmentcapable of specific binding to a polypeptide having at least 67%sequence similarity with SEQ ID NO.:153 and of impairing an activity ofthe polypeptide in osteoclast precursor cells or in osteoclasts and; b.A pharmaceutically acceptable carrier.
 71. The pharmaceuticalcomposition of claim 70, wherein the polypeptide has a sequence at least80% identical, at least 90% identical or at least 95% identical to SEQID NO.:153 or SEQ ID NO.:93.
 72. The pharmaceutical composition of claim70, wherein the polypeptide has a sequence identical to SEQ ID NO.:153or SEQ ID NO.:93.
 73. The pharmaceutical composition of claim 70,wherein the antibody or antigen binding fragment inhibits osteoclastdifferentiation.
 74. The pharmaceutical composition of claim 70, whereinthe antibody is selected from the group consisting of a polyclonalantibody, a monoclonal antibody, a chimeric antibody, a humanizedantibody and a human antibody.
 75. The pharmaceutical composition ofclaim 70, wherein the antigen binding fragment is a FV, a Fab, a Fab′ ora (Fab′)₂.
 76. The pharmaceutical composition of claim 70, furthercomprising a drug or an hormone.
 77. The pharmaceutical composition ofclaim 76, wherein the drug is an antiresorptive drug or a drugincreasing bone mineral density.
 78. The pharmaceutical composition ofclaim 70, wherein the antibody or antigen binding fragment is conjugatedwith a therapeutic agent.